Vaccines and compositions against Streptococcus pneumoniae

ABSTRACT

Streptococcus pneumoniae  is a major health concern, especially in very young, elderly, or immunocompromized patients. The present disclosure provides, inter alia, certain highly effective vaccines and pharmaceutical compositions in  Streptococcus pneumoniae . The antigens may be used therapeutically or prophylactically.

RELATED APPLICATION

This application claims the benefit of the filing date of U.S.Provisional Application No. 61/434,818, filed Jan. 20, 2011. The entireteachings of the referenced application are expressly incorporatedherein by reference.

GOVERNMENT SUPPORT

This work was made with Government support under Grant AI066013 awardedby the National Institutes of Health. Therefore, the U.S. Government hascertain rights in this invention.

I. BACKGROUND

Pneumococcal disease continues to be a leading cause of sickness anddeath in the United States and throughout the world. Each year, millionsof cases of pneumonia, meningitis, bacteremia, and otitis media areattributed to infection with the pathogen Streptococcus pneumoniae. S.pneumoniae is a Gram-positive encapsulated coccus that colonizes thenasopharynx in about 5-10% of healthy adults and 20-40% of healthychildren. Normal colonization becomes infectious when S. pneumoniae iscarried into the Eustachian tubes, nasal sinuses, lungs, bloodstream,meninges, joint spaces, bones and peritoneal cavity. S. pneumoniae hasseveral virulence factors that enable the organism to evade the immunesystem. Examples include a polysaccharide capsule that preventsphagocytosis by host immune cells, proteases that inhibitcomplement-mediated opsonization, and proteins that cause lysis of hostcells. In the polysaccharide capsule, the presence of complexpolysaccharides forms the basis for dividing pneumococci into differentserotypes. To date, 93 serotypes of S. pneumoniae have been identified.

Various pharmaceutical compositions have been used to harness an immuneresponse against infection by S. pneumoniae. A polyvalent pneumococcalvaccine, PPV-23, was developed for preventing pneumonia and otherinvasive diseases due to S. pneumoniae in the adult and agingpopulations. The vaccine contains capsular polysaccharides (CPs) from 23serotypes of S. pneumoniae. As T cell-independent antigens, these CPsinduce only short-lived antibody responses, necessitating repeateddoses, which increases the risk of immunological tolerance. Theantibodies raised against S. pneumoniae, termed anticapsular antibodies,are recognized as protective in adult and immunocompetent individuals.However, children under 2 years of age and immunocompromisedindividuals, including the elderly, do not respond well to Tcell-independent antigens and, therefore, are not afforded optimalprotection by PPV-23. Another S. pneumoniae vaccine, Prevnar, includesbacterial polysaccharides from 7 S. pneumoniae strains conjugated to thediphtheria toxoid protein. This vaccine induces both B and T cellresponses. However, because it only protects against 7 pneumococcalserotypes, serotype replacement can render Prevnar ineffective. Serotypereplacement has already been demonstrated in several clinical trials andepidemiologic studies, necessitating development of differentformulations of these vaccines. An example is the recently introducedPrevnar 13, directed to 13 pneumococcal serotypes. Furthermore, the twoPrevnar formulations are expensive to manufacture, greatly limitingtheir availability in the developing world. PPV-23, which consists of 23purified but unconjugated polysaccharides, has broader coverage, butdoes not provide protection to children under the age of 2 years, apopulation which is at the highest risk for pneumococcal disease.

Thus, there remains a need to design more effective pharmaceuticalcompositions than the current strategies offer. In particular, suchcompositions need to incorporate novel or specific antigens that elicitan immune response against S. pneumoniae.

II. SUMMARY

Streptococcus pneumoniae is a major health concern, especially in veryyoung, elderly, or immunocompromised patients. While DNA and proteinsequence information for S. pneumoniae has been known for some time, andresearchers have long attempted to produce vaccines against S.pneumoniae, a major problem was how to identify protective polypeptidesfrom among the approximately 2100 genes in the S. pneumoniae genome. Theinstant application presents the results of whole-genome screensdesigned to identify the most immunogenic proteins in the S. pneumoniaegenome. Several of the hits from the screen have been shown to protectagainst S. pneumoniae colonization in a mouse model, and in someinstances against both colonization and S. pneumoniae-induced sepsis.Accordingly, the present disclosure provides, inter alia, certain highlyeffective vaccines against Streptococcus pneumoniae. The vaccines may beused therapeutically or prophylactically. The present disclosure alsoprovides specific antigens and methods for using the antigens to elicitan immune response against S. pneumoniae.

In certain aspects, the present disclosure provides a vaccineformulation comprising a pharmaceutically acceptable carrier and apolypeptide having an amino acid sequence comprising (or consisting of)SEQ ID NO: 265 or 268 or an immunogenic fragment thereof. In someembodiments, the polypeptide comprises an exogenous signal sequence. Forinstance, the polypeptide may have an amino acid sequence comprising SEQID NO: 266 or an immunogenic fragment thereof. The polypeptide may havean amino acid sequence consisting of SEQ ID NO: 265, 266, or 268.

In some embodiments, the vaccine formulation further comprises a firstpolypeptide having an amino acid sequence comprising (or consisting of)one of SEQ ID NOS: 1-23, 267, and 269-270 or an immunogenic fragmentthereof. In certain embodiments, the vaccine formulation furthercomprises a second polypeptide having an amino acid sequence comprisingany of SEQ ID NOS: 1-23, 267, and 269-270 or an immunogenic fragmentthereof.

In certain embodiments, the first and the second polypeptides belong toa different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenicfragment thereof, (ii) one of SEQ ID NOS: 2-5 and 14-17 or animmunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 and 18-19 oran immunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenicfragment thereof, (v) one of SEQ ID NOS: 9-10 and 20-21 or animmunogenic fragment thereof, and (vi) one of SEQ ID NO: 11-13, 267, and269-270 or an immunogenic fragment thereof.

In some such embodiments, the vaccine formulation comprises apolypeptide having an amino acid sequence comprising SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, thevaccine formulation comprises a polypeptide consisting of SEQ ID NO: 6or 7 and a polypeptide consisting of SEQ ID NO: 9 or 10.

In any of the aspects or embodiments herein, the vaccine formulation maycomprise a polypeptide of SEQ ID NO: 265, 266, or 268 which is atruncated fragment having from 1-20 amino acid residues removed from theN-terminus, C-terminus, or both. In some embodiments, the vaccineformulation contains substantially no other S. pneumoniae polypeptidesother than polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 1-21 and 265-270.

In certain embodiments, the vaccine formulation comprises one or morepolypeptides having an amino acid sequence comprising SEQ ID NOS: 22 or23 or an immunogenic fragment thereof.

In another aspect, the invention provides vaccine formulationscomprising a known S. pneumoniae antigen, such as a pneumolysoid,Choline-binding protein A (CbpA), or Pneumococcal surface protein A(PspA), or derivatives thereof, and one, two, or three polypeptides fromTable 1 or Table 2. An exemplary vaccine formulation comprises: (i) apolypeptide having an amino acid sequence comprising (or consisting of)one or more of SEQ ID NOS: 1-23 and 265-270 or an immunogenic fragmentthereof, (ii) a pneumolysoid, and (iii) a pharmaceutically acceptablecarrier. A further exemplary vaccine formulation comprises: (i) apolypeptide having an amino acid sequence comprising (or consisting of)one or more of SEQ ID NOS: 1-23 and 265-270 or an immunogenic fragmentthereof, (ii) CbpA or a derivative thereof, and (iii) a pharmaceuticallyacceptable carrier. A further exemplary vaccine formulation comprises:(i) a polypeptide having an amino acid sequence comprising (orconsisting of) one or more of SEQ ID NOS: 1-23 and 265-270 or animmunogenic fragment thereof, (ii) PspA or a derivative thereof, and(iii) a pharmaceutically acceptable carrier. In some such embodiments,the polypeptide of (i) comprises any one of SEQ ID NO: 2-5, 6, 7, 9-13,and 265-267. In some embodiments, the vaccine formulation furthercomprises a second polypeptide having an amino acid sequence comprisingone of SEQ ID NO: 1-23 and 265-270. In some embodiments, thepneumolysoid is PdT, Pd-A, Pd-B, rPd2, rPd3, Ply8, Δ6PLY, L460D (see,e.g., US 2009/0285846 and L. Mitchell, Protective Immune Responses toStreptococcus pneumoniae Pneumolysoids, ASM2011 conference abstract,2011), or a variant thereof. In some embodiments, the derivative of PspAcomprises all or a fragment of the proline-rich region of PspA.

In certain embodiments, the polypeptide is conjugated to an immunogeniccarrier. In some embodiments, the vaccine formulation comprises at leastone lipidated polypeptide.

In some embodiments, the vaccine formulation further comprisesconjugated S. pneumoniae polysaccharides. The conjugated polysaccharidesmay be, for example, as described in U.S. Pat. No. 5,623,057, U.S. Pat.No. 5,371,197, or PCT/US2011/023526.

In some embodiments, the vaccine formulation further comprises anadjuvant. The adjuvant may be, for example, an agonist of toll-likereceptors (TLRs). The adjuvant may be, for example, alum. In someembodiments, the vaccine formulation comprises 1-1000 μg of eachpolypeptide and 1-250 μg of the adjuvant.

In certain embodiments, the vaccine formulation induces a T_(H)17 cellresponse at least 1.5-fold greater than that induced by a controlunrelated antigen after contacting T_(H)17 cells. In some embodiments,the vaccine formulation inhibits infection by S. pneumoniae in anuninfected subject. In certain embodiments, the vaccine formulationinhibits S. pneumoniae colonization in an individual. In someembodiments, the vaccine formulation inhibits S. pneumoniae symptoms orsequelae. For instance, the vaccine formulation inhibits S.pneumoniae-induced sepsis.

In certain aspects, the present disclosure provides a method fortreating a subject suffering from or susceptible to S. pneumoniaeinfection, comprising administering an effective amount of any of thevaccine formulations described herein.

In some embodiments, the method inhibits infection by S. pneumoniae inan uninfected subject. In some embodiments, the method inhibits S.pneumoniae colonization in a subject. In some embodiments, the methodinhibits S. pneumoniae symptoms or sequelae. An exemplary sequela issepsis.

In certain embodiments, the method treats a subject with one dose. Inother embodiments, the method treats a subject with two or three doses.In some embodiments, the method treats a subject within three doses.

In certain embodiments, the subject is a human.

The present disclosure provides, for example, a vaccine formulationcomprising a pharmaceutically acceptable carrier and one or morepolypeptides having an amino acid sequence comprising any of SEQ ID NOS:1-13, 265, 266 and 267, or an immunogenic fragment thereof.

The present disclosure also provides a vaccine formulation comprising apharmaceutically acceptable carrier and at least one polypeptide havingan amino acid sequence comprising SEQ ID NO: 6, SEQ ID NO:10 or SEQ IDNO: 265, or an immunogenic fragment thereof. The present disclosurefurther provides a vaccine formulation comprising a pharmaceuticallyacceptable carrier and at least one polypeptide having an amino acidsequence consisting of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 265, oran immunogenic fragment thereof.

Furthermore, the instant application provides a vaccine formulationcomprising a pharmaceutically acceptable carrier and one or morepolypeptides having an amino acid sequence comprising any of SEQ ID NOS:14-23, 268, 269 and 270, or an immunogenic fragment thereof.

The present disclosure further provides an immunogenic compositioncomprising a pharmaceutically acceptable carrier and two or morepolypeptides having amino acid sequences comprising any of SEQ ID NOS:1-13, 265, 266 and 267, or an immunogenic fragment thereof.

The present disclosure also provides a vaccine formulation comprising apharmaceutically acceptable carrier and two or more polypeptides havingamino acid sequences comprising SEQ ID NO: 6, SEQ ID NO:10 or SEQ ID NO:265, or an immunogenic fragment thereof. The present disclosure furtherprovides a vaccine formulation comprising a pharmaceutically acceptablecarrier and two or more polypeptides having amino acid sequencesconsisting of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 265, or animmunogenic fragment thereof. This disclosure also provides a vaccineformulation comprising a pharmaceutically acceptable carrier and two ormore polypeptides having amino acid sequences comprising SEQ ID NO: 6,SEQ ID NO: 9, and SEQ ID NO: 265, or an immunogenic fragment thereof. Inaddition, this disclosure provides a vaccine formulation comprising apharmaceutically acceptable carrier and two or more polypeptides havingamino acid sequences comprising SEQ ID NO: 7, SEQ ID NO: 10, and SEQ IDNO: 265, or an immunogenic fragment thereof. In some embodiments, theamino acid sequence comprising SEQ ID NO: 265 comprises an exogenoussignal sequence.

The present disclosure also provides a vaccine formulation comprising apharmaceutically acceptable carrier and three or more polypeptideshaving amino acid sequences comprising SEQ ID NO: 6, SEQ ID NO:10 andSEQ ID NO: 265, respectively, or an immunogenic fragment thereof. Thepresent disclosure further provides a vaccine formulation comprising apharmaceutically acceptable carrier and three or more polypeptideshaving amino acid sequences consisting of SEQ ID NO: 7, SEQ ID NO: 9 andSEQ ID NO: 265, respectively, or an immunogenic fragment thereof.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and cholera toxinadjuvant, then stimulated with killed, unencapsulated whole cell S.pneumoniae, as described in Example 5. The left panel shows the data inscatter format, and the right panel shows the average and standarddeviation for each sample. Immunization group “All 3” represents animalsimmunized with a combination of SP2108, SP0148, and SP1634.

FIG. 2 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and cholera toxinadjuvant, then stimulated with a combination of three proteins (SP2108,SP0148, and SP1634), as described in Example 5.

FIG. 3 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated protein(s) andcholera toxin adjuvant, then challenged with intranasal administrationof S. pneumoniae, as described in Example 5. 003 represents a controlunrelated antigen.

FIG. 4 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and cholera toxinadjuvant, then stimulated with killed, unencapsulated whole cell S.pneumoniae, as described in Example 6.

FIG. 5 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and cholera toxinadjuvant, then stimulated by the indicated protein(s), as described inExample 6.

FIG. 6 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated protein(s) andcholera toxin adjuvant, then challenged with intranasal administrationof S. pneumoniae, as described in Example 6. The HSV-2 protein ICP47with the gene name US12 (NP_044543.1, NC_001798.1; shown in the figureas 003) and ovalbumin (OVA) represent control antigens.

FIG. 7 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated protein(s) andcholera toxin adjuvant, then challenged with intranasal administrationof S. pneumoniae, as described in Example 7.

FIG. 8 shows the number of S. pneumoniae colonies obtained from a nasalwash in BALB/c mice that were immunized with the indicated protein(s)and cholera toxin adjuvant, then challenged with intranasaladministration of S. pneumoniae, as described in Example 8.

FIG. 9 shows the concentration of IL-17A generated by blood samples frommice that were immunized with the indicated proteins and cholera toxinadjuvant, then stimulated with the protein of immunization (left panel)or killed, unencapsulated whole cell S. pneumoniae (right panel), asdescribed in Example 9.

FIG. 10 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated proteins and choleratoxin adjuvant, then challenged with intranasal administration of S.pneumoniae, as described in Example 10.

FIG. 11 shows survival of mice that were immunized with the indicatedproteins and the adjuvant alum, then underwent aspiration challenge withS. pneumoniae as described in Example 11.

FIG. 12 shows survival of mice that were immunized with the indicatedproteins and the adjuvant alum, then underwent aspiration challenge withS. pneumoniae as described in Example 12.

FIG. 13 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated proteins and choleratoxin adjuvant, then challenged with intranasal administration of S.pneumoniae, as described in Example 13.

FIG. 14 shows the concentration of IL-17A generated by blood samplesform mice that were immunized with the indicated proteins and alum, thenstimulated with the proteins indicated at upper left, as described inExample 14.

FIG. 15 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated proteins and alum orwith killed, unencapsulated whole cell S. pneumoniae plus alum (WCV),then challenged with intranasal administration of S. pneumoniae, asdescribed in Example 15.

FIG. 16 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated proteins and alum orwith killed, unencapsulated whole cell S. pneumoniae plus alum (WCV),then challenged with intranasal administration of S. pneumoniae, in twopooled studies as described in Example 16.

FIG. 17 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated proteins and alum orwith killed, unencapsulated whole cell S. pneumoniae plus alum (WCB),then challenged with intranasal administration of S. pneumoniae, asdescribed in Example 17.

FIG. 18 shows survival of mice that were injected with antibodies orsera specific to the indicated proteins, then underwent aspirationchallenge with S. pneumoniae, as described in Example 18.

FIG. 19 shows the percent of animals protected from sepsis in sixseparate aspiration challenge studies, two of which are described inmore detail in Examples 12 and 18.

IV. DETAILED DESCRIPTION

A. Specific Polypeptides and Nucleic Acids for Use in S. pneumoniaeVaccines and Immunogenic Compositions

This application describes S. pneumoniae vaccines that include one ormore of the polypeptides or genes listed in Table 1, or variants orfragments thereof as described below. The vaccine may include apolypeptide that comprises a sequence of Table 1 or a variant orimmunogenic fragment thereof or a polypeptide that consists of asequence of Table 1 or a variant or immunogenic fragment thereof. TheDNA and protein sequence of each gene and polypeptide may be found bysearching for the Locus Tag in the publicly available database, EntrezGene (on the NCBI NIH web site on the World Wide Web, atwww.ncbi.nlm.nih.gov/sites/entrez?db=gene), in the Streptococcuspneumoniae TIGR4 genome, and the indicated sequences are also includedin this application.

TABLE 1 Immunogenic polypeptides for vaccine formulations DNA GenBankProtein DNA Accession No. Locus tag name and description SEQ ID No. SEQID No. (from Mar. 30, 2010) SP0024 1 — NC_003028.3|: 27381-27878 SP08822 — NC_003028.3|: 831804-832628 SP0882N 3 24 — SP0882 with exogenoussignal sequence 4 25 — SP0882N with exogenous signal sequence 5 26 —SP0148 lacking signal sequence 6 27 — SP0148 including signal sequence 728 NC_003028.3|: 145,513-146,343* SP1072 8 — NC_003028.3|:1008420-1010180 SP2108 including signal sequence 9 — NC_003028.3|:2020750-2022021 SP2108 lacking signal sequence 10 29 — SP0641M 11 30 —SP0641 12 — NC_003028.3|: 2020750-2022021 SP0641N 13 31 — SP0882consensus 14 — — SP0882N consensus 15 — — SP0882 consensus withexogenous leader 16 — — SP0882N consensus with exogenous leader 17 — —SP0148 consensus lacking signal sequence 18 — — SP0148 consensusincluding signal sequence 19 — — SP2108 consensus lacking signalsequence 20 — — SP2108 consensus including signal sequence 21 — — SP163422 — NC_003028.3|: 1534348-1535421 SP0314 23 — NC_003028.3|:287483-290683 SP1912 265 271 NC_003028.3|: 1824672-1824971 SP1912L 266272 — SP0641.1 267 273 — SP1912 consensus 268 — — SP0641N consensus 269— — SP0641M consensus 270 — — *NB: The database sequence incorrectlylists TTG (encoding Leu) at nucleotide positions 541-543. The correctsequence, as shown in SEQ ID NO: 28, has TTC at that codon and encodesPhe. The database sequence further does not include a C-terminal Glufound in certain isolates.

Certain polypeptides of Table 1, and variants thereof, are described ingreater detail below.

1. SP1912 (SEQ ID NO: 265) and Variants Thereof

SP1912 is a hypothetical protein of 99 amino acids. While the proteinfunction is not definitively known, sequence analysis suggests it is aputative thioredoxin.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP1912. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 90, 75, 60, 45 or 30consecutive amino acids from SP1912.

In some embodiments, the compositions and methods herein call for theuse of an SP1912 variant that comprises an exogenous lipidationsequence. In some embodiments, a signal sequence directs lipidation.Thus, the lipidation signal may be, e.g., the signal sequence of SP2108(SEQ ID NO: 275) or SP0148, or an E. coli signal sequence. The exemplaryvariant SP1912L, comprising the signal sequence of the E. coli gene RlpB(SEQ ID NO: 276) is represented by polypeptide sequence SEQ ID NO: 266.SP1912 (SEQ ID NO: 265) and SP1912L (SEQ ID NO: 266) may be encoded,respectively, by nucleic acids according to SEQ ID NO: 271 and 272,although due to degeneracy in the genetic code, other DNA sequences(including codon-optimized sequences) may be used.

Consensus sequences illustrating combinations of SP1912 sequences fromdifferent serotypes are provided as SEQ ID NO: 268. Thus, in certainembodiments, the vaccine formulation comprises a polypeptide having anamino acid sequence comprising, or consisting of, SEQ ID NO: 268, or animmunogenic fragment thereof (e.g., in place of a polypeptide having anamino acid sequence comprising SEQ ID NO: 265).

2. SP0024 (SEQ ID NO: 1) and Variants Thereof

SP0024 represents a hypothetical protein of 165 amino acids, containinga conserved carbonic anhydrase domain that extends from amino acid 27 toamino acid 163. Based on this consensus motif, SP0024 may be azinc-binding protein.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0024. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 150, 125, or 100consecutive amino acids from SP0024.

3. SP0882 (SEQ ID NO: 2) and Variants Thereof

SP0882 is a conserved hypothetical protein of 274 amino acids. Much ofthe protein (amino acids 2-270) forms an esterase or lipase-like region.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0882. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 250, 275, 200, 175,150, 125, or 100 consecutive amino acids from SP0882.

One particular truncation variant named SP0882N consists of theN-terminal 130 amino acids of SP0882, and is shown as SEQ ID NO: 3.SP0882N includes a region that is particularly well conserved amongdifferent serotypes. In certain embodiments, a polypeptide comprisingSP0882 or SP0882N, or an immunogenic fragment of either, also comprisesan exogenous signal sequence. In some embodiments, the signal sequenceis an E. coli or S. pneumoniae signal sequence. The signal sequence maybe, for example, the signal sequence of SP2108. Two exemplary suchpolypeptides are SEQ ID NOS: 4 and 5.

Variants of DNA and protein sequences of SP0882 are described, interalia, in US Patent Application Publication No. 2009/0215149 andInternational Applications WO2002/077021, WO98/18931, and WO2007/106407.A variant of SP0882N is disclosed in International ApplicationWO2008/146164.

Sequence variation occurs at the protein level between different S.pneumoniae serotypes, and consensus sequences illustrating combinationsof SP0882 sequences from different S. pneumoniae serotypes are providedas SEQ ID NOS: 14-17. Accordingly, in certain embodiments, the vaccineformulation comprises a polypeptide having an amino acid sequencecomprising, or consisting of, any of SEQ ID NOS: 14-17, or animmunogenic fragment thereof (e.g., in place of a polypeptide having anamino acid sequence comprising one of SEQ ID NOS: 2-5).

Nucleic acid sequences encoding different variants of SP0882 (SEQ IDNOS: 2-5) are provided as SEQ ID NOS: 24-26, although due to degeneracyin the genetic code, other DNA sequences (including codon-optimizedsequences) could encode these polypeptides.

4. SP0148 (SEQ ID NO: 7) and Variants Thereof.

The protein SP0148 is named “ABC transporter, substrate-bindingprotein”. Proteins of this class are typically extracellular proteinsthat interact transiently with a transmembrane protein complex. Suchcomplexes use energy generated by ATP hydrolysis to translocate specificsubstrates across a cell membrane. SP0148 is a 276 or 277 (depending onthe isolate) amino acid protein that contains a conserved PBPb(periplasmic binding protein) domain, spanning amino acids 40-246, whichis typical of membrane-bound transport complexes. In addition, SP0148has a bacterial extracellular solute-binding proteins family 3 domainwhich is largely co-extensive with the PBPb domain and extends fromamino acid 40 to 244. In some embodiments, a vaccine or othercomposition comprises a truncation mutant of SP0148 comprising orlacking one or more of said domains and motifs.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0148. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 250, 275, 200, 175,150, 125, or 100 consecutive amino acids from SP0148.

Endogenous SP0148 comprises a signal sequence that directs its secretionand potential lipidation. In some embodiments, the signal sequence ofthe polypeptide of SEQ ID NO: 7 is partially or fully processed by anexpression host, e.g. E. coli. In some embodiments, a variant of SP0148that lacks the signal sequence (SEQ ID NO: 6) is used. The polypeptideof SEQ ID NO: 6 is encoded by the nucleic acid of SEQ ID NO: 27,although other nucleic acid sequences (including codon-optimizedsequences) may be used. SEQ ID NO: 28 encodes the full length sequenceof SP0148 used in the screens herein.

Variants of the amino acid sequence and nucleotide sequence of SP0148may be found in U.S. Patent Application Publication No. 2005/0020813,U.S. Pat. Nos. 7,378,514 and 7,504,110, and European Patent ApplicationNo. EP1572868 and EP1855717.

Consensus sequences illustrating combinations of SP0148 sequences fromdifferent S. pneumoniae serotypes are provided as SEQ ID NOS: 18 and 19.Accordingly, in certain embodiments, the vaccine formulation comprises apolypeptide having an amino acid sequence comprising, or consisting of,either of SEQ ID NOS: 18-19, or an immunogenic fragment thereof (e.g.,in place of a polypeptide having an amino acid sequence comprising oneof SEQ ID NOS: 6 or 7).

5. SP1072 (SEQ ID NO: 8) and Variants Thereof

SP1072, also known as dnaG, is a DNA primase enzyme that catalyzesformation of an RNA primer which allows DNA polymerase to initiate DNAreplication. A protein of 586 amino acids, SP1072 contains severalconserved motifs. Beginning at the N-terminus, amino acids 2-96 form azinc finger domain, the DNA primase catalytic core spans amino acids122-250, and a highly conserved topoisomerase-primase (TORPIM)nucleotidyl transferase/hydrolase domain region extends from amino acid258 to 330. In some embodiments, a vaccine or other compositioncomprises a truncation mutant of SP1072 comprising or lacking one ormore of said domains and motifs.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected form SP1072. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 550, 500, 450, 400,350, 300, 250, 200, 150, or 100 consecutive amino acids from SP1072.

6. SP2108 (SEQ ID NO: 9) and Variants Thereof.

The polypeptide SP2108 is 423 amino acids in length and is alternativelyknown as MalX, maltose/maltodextrin ABC transporter, ormaltose/maltodextrin-binding protein. Much of the protein (amino acids3-423) is classified as a MalE (Maltose-binding periplasmic) domain. Inaddition, SP2108 contains a signal sequence that directs its secretionand potential lipidation. In some embodiments, the signal sequence ofthe polypeptide of SEQ ID NO: 9 is partially or fully processed by anexpression host, e.g. E. coli. In some embodiments, a vaccine or othercomposition comprises a truncation mutant of SP2108 comprising one ormore of said domains and motifs.

In some embodiments, the compositions and methods herein call for theuse of an SP2108 variant that lacks the signal sequence. This variant isrepresented by polypeptide sequence SEQ ID NO: 10 and may be encoded by,for example, a nucleic acid according to SEQ ID NO: 29, although due todegeneracy in the genetic code, other DNA sequences (includingcodon-optimized sequences) may be used.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP2108. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 400, 350, 300, 250,200, 150, or 100 consecutive amino acids from SP2108.

Consensus sequences illustrating combinations of SP2108 sequences fromdifferent serotypes are provided as SEQ ID NOS: 20 and 21. Thus, incertain embodiments, the vaccine formulation comprises a polypeptidehaving an amino acid sequence comprising, or consisting of, either ofSEQ ID NOS: 20-21, or an immunogenic fragment thereof (e.g., in place ofa polypeptide having an amino acid sequence comprising one of SEQ IDNOS: 9 or 10).

7. SP0641 (SEQ ID NO: 12) and Variants Thereof.

At 2144 amino acids in length, SP0641 is also known as PrtA, a cellwall-associated serine protease. Full-length SP0641 contains a number ofconserved motifs: the PA_2 motif, extending between amino acids 485 and597, which may form a protein binding surface; the Fn3-like domain(amino acids 800-939); and two predicted catalytic domains of the S8 C5atype located at amino acids 226-449 and 639-777. In some embodiments, avaccine or other composition comprises a truncation mutant of SP0641comprising or lacking one or more of said domains and motifs.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0641. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 1000, 900, 800, 700,600, 500, 400, 300, 200, or 100 consecutive amino acids from SP0641.

Certain other truncation mutants of SP0641 may also be used. Forinstance, the polypeptide designated SP0641N (SEQ ID NO: 13) consists of661 amino acids corresponding to amino acids 24-684 near the N-terminusof SP0641. Roughly adjacent to SP0641N (and corresponding to amino acids686-1333 of SP0641) lies the 648 residue region captured by thetruncation variant SP0641M (SEQ ID NO: 11). The polypeptide designatedSP0641.1 (SEQ ID NO: 267) consists of 978 amino acids corresponding toamino acids 28-1006 of SP0641.

Variants of SP0641 are disclosed in, for example, U.S. Pat. Nos.7,338,786, 6,573,082, and 7,132,107, as well as InternationalApplication WO00/06738.

SEQ ID NOS: 30, 31 and 273 display the DNA sequences of SP0641M (SEQ IDNO: 11), SP0641N (SEQ ID NO: 13) and SP641.1 (SEQ ID NO: 267),respectively, although due to degeneracy in the genetic code, other DNAsequences (including codon-optimized sequences) could encode theseSP0641 variants.

Consensus sequences illustrating combinations of SP0641N and SP0641Msequences from different S. pneumoniae serotypes are provided as SEQ IDNOS: 269 and 270. Accordingly, in certain embodiments, the vaccineformulation comprises a polypeptide having an amino acid sequencecomprising, or consisting of, either of SEQ ID NOS: 269 or 270, or animmunogenic fragment thereof (e.g., in place of a polypeptide having anamino acid sequence comprising one of SEQ ID NOS: 11 or 13).

Polypeptides homologous to the polypeptides of Tables 1 and 2 (forexample, SP1912, SP1912L, SP0024, SP0882, SP0882N, SP0148 with orwithout a signal sequence, SP1072, SP2108 with or without a signalsequence, SP0641, SP0641M, SP0641N, or SP0641.1) may also be used in thecompositions and methods disclosed herein. Individual strains of S.pneumoniae contain numerous mutations relative to each other, and someof these result in different protein sequences between the differentstrains. One of skill in the art may readily substitute an amino acidsequence, or a portion thereof, with the homologous amino acid sequencefrom a different S. pneumoniae strain. In certain aspects, thisapplication provides immunogenic polypeptides with at least 90%, 95%,97%, 98%, 99%, or 99.5% identity to the polypeptides of Tables 1 and 2or an immunogenic fragment thereof. Serotypic variation may be used todesign such variants of the polypeptides of Tables 1 and 2.

In some embodiments, the vaccine compositions herein comprise a fragmentof a protein of Table 1 or 2 (for example, fragments of SP1912, SP1912L,SP0024, SP0882, SP0882N, 0SP148 with or without a signal sequence,SP1072, SP2108 with or without a signal sequence, SP0641, SP0641M,SP0641N, or SP0641.1). In some embodiments, this application providestruncation mutants that are close in size to the polypeptide of Table 1or 2 (for example, one of SEQ ID NOS: 1-13, 265, 266 or 267). Forexample, they may lack at most one, two three, four, five, ten, ortwenty amino acids from one or both termini. Internal deletions, e.g.,of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated.

In certain embodiments the vaccine formulation comprises one or morepolypeptides having an amino acid sequence comprising, or consisting of,any of SEQ ID NOS: 14-21, 268, 269 and 270. In certain embodiments, thefragment is a truncated fragment of any of SEQ ID NOS: 14-21, 268, 269and 270, wherein from 1-5, 1-10, or 1-20 amino acid residues are removedfrom the N-terminus, C-terminus, or both. In certain embodiments, thefragment is a truncated fragment of any of SEQ ID NOS: 14-21, 268, 269and 270, wherein from 1-10 amino acid residues are removed from theN-terminus, C-terminus, or both. For instance, 10 amino acid residuesmay be removed from each of the N-terminus and C-terminus resulting in aprotein with 20 amino acid residues removed.

In certain embodiments, the vaccine formulations provided hereincomprise or further comprise one or more, or two or more, known S.pneumoniae antigens. In some instances, the known S. pneumoniae antigensare predominantly antibody targets. In some instances, the known S.pneumoniae antigens protect from S. pneumoniae colonization, or from S.pneumoniae-induced sepsis. One appropriate art-recognized class of S.pneumoniae antigen is the pneumolysoids. Pneumolysoids have homology tothe S. pneumoniae protein pneumolysin (PLY), but have reduced toxicitycompared to pneumolysin. Pneumolysoids can be naturally occurring orengineered derivatives of pneumolysin. In some embodiments, apneumolysoid has at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identity to pneumolysin. In some embodiments, the pneumolysoiddemonstrates less than 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500,or 1/1000 the toxicity of pneumolysin in an assay for one or both ofhemolytic activity towards erythrocytes and inhibition ofpolymorphonuclear leukocytes. Both assays are described in Saunders F.K. et al. (“Pneumolysin, the thiol-activated toxin of Streptococcuspneumoniae, does not require a thiol group for in vitro activity” InfectImmun. 1989 August; 57 (8):2547-52).

Exemplary pneumolysoids include PdT (a triple mutant further describedin Berry, A. M. et al. (1995) Infection and Immunity 63:1969-74); Pd-Aand Pd-B (Paton J. C. et al. “Purification and immunogenicity ofgenetically obtained pneumolysin toxoids and their conjugation toStreptococcus pneumoniae type 19F polysaccharide” Infect Immun. 1991July; 59 (7):2297-304); rPd2 and rPd3 (Ferreira et al. “DNA vaccinesbased on genetically detoxified derivatives of pneumolysin fail toprotect mice against challenge with Streptococcus pneumoniae” FEMSImmunol Med Microbiol (2006) 46: 291-297); Ply8, Δ6PLY, L460D, or avariant thereof. In some embodiments, the pneumolysin has a mutation inthe catalytic center, such as at amino acid 428 or 433 or the vicinity.

Other appropriate S. pneumoniae antigens for combination vaccinesinclude Pneumococcal surface protein A (PspA); derivatives of PspA,Choline-binding protein A (CbpA) and derivatives thereof (AD Ogunniyi etal., “Protection against Streptococcus pneumoniae elicited byimmunization with pneumolysin and CbpA,” Infect Immun. 2001 October; 69(10):5997-6003); Pneumococcal surface adhesin A (PsaA); caseinolyticprotease; sortase A (SrtA); pilus 1 RrgA adhesin; PpmA; PrtA; PavA;LytA; Stk-PR; PcsB; RrgB and derivatives thereof.

Derivatives of PspA include proline-rich segments with the non-prolineblock (PR+NPB, further described below as well as in Daniels, C. C. etal. (2010) Infection and Immunity 78:2163-72) and related constructscomprising all or a fragment of the proline-rich region of PspA (e.g.,regions containing one or more of the sequences PAPAP, PKP, PKEPEQ andPEKP and optionally including a non-proline block). An example of thenon-proline-block has the exemplary sequence EKSADQQAEEDYARRSEEEYNRLTQQQ(SEQ ID NO: 306), which generally has no proline residues in anotherwise proline-rich area of the non-coiled region of PspA. Otherembodiments of non-proline block (NPB) sequences include SEQ ID NOs: 307and 308. PspA and its derivatives can include genes expressing similarproline-rich structures (i.e. PKP, PKEPEQ and PEKP), with or without theNPB. The amino acids at either end of the NPB mark the boundaries of theproline-rich region. In one example, the amino-terminal boundary to thePR-region is DLKKAVNE (SEQ ID NO: 309), and the carboxy-terminalboundary is (K/G)TGW(K/G)QENGMW (SEQ ID NO: 310). Peptides containingthe NPB are particularly immunogenic, suggesting that the NPB may be animportant epitope. Exemplary immunogenic PspA polypeptide derivativescontaining the coiled-coil structure include SEQ ID NOs: 301 and 302.Particular embodiments of the immunogenic PspA polypeptide derivativeslacking the coiled-coil structure have the amino acid sequences shown asSEQ ID NOS: 303-305. Immunogenic PspA polypeptides SEQ ID NO: 301, 303and 305 include both PR and NPB sequences (PR+NPB). Immunogenic PspApolypeptides of SEQ ID NOS: 302 and 304 include only a PR sequence (PRonly) and lack the NPB.

In some cases, the other appropriate S. pneumoniae antigen is at leastat least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to thecorresponding wild-type S. pneumoniae protein. Sequences of theabove-mentioned polypeptides, and nucleic acids that encode them, areknown; see, for example, the S. pneumoniae ATCC 700669 complete genomesequence under GenBank accession number FM211187.1 and linkedpolypeptide sequences therein.

Further S. pneumoniae antigens for combination vaccines includeconjugated S. pneumoniae polysaccharides. The conjugated polysaccharidesmay be, for example, as described in U.S. Pat. No. 5,623,057, U.S. Pat.No. 5,371,197, or PCT/US2011/023526.

In addition to those nucleic acids and polypeptides described in Table 1above, this application also provides immunogenic compositions thatinclude one or more of the polypeptides or genes listed in Table 2, orvariants or fragments thereof as described herein. The DNA and proteinsequence of each gene and protein may be found by searching for theLocus Tag in the publicly available database, Entrez Gene, as describedabove.

TABLE 2 Immunogenic proteins identified in human and mouse screens Locustag Protein accession DNA accession number (from name number Mar. 30,2010) SP1574 AAK75660.1 NC_003028.3|: c1481367-1480609 SP1655 AAK75734.1NC_003028.3|: c1557922-1557230 SP2106 AAK76165.1 NC_003028.3|:c2018657-2016399 SP1473 AAK75567.1 NC_003028.3|: c1386534-1386277 SP0605AAK74757.1 NC_003028.3|: 571604-572485 SP1177 AAK75286.1 NC_003028.3|:c1115580-1115317 SP0335 AAK74510.1 NC_003028.3|: 306559-306876 SP0906AAK75031.1 NC_003028.3|: c859160-859029 SP1828 AAK75901.1 NC_003028.3|:c1740010-1739000 SP2157 AAK76211.1 NC_003028.3|: c2072146-2070995 SP1229AAK75335.1 NC_003028.3|: c1163388-1161718 SP1128 AAK75238.1NC_003028.3|: 1061773-1063077 SP1836 AAK75909.1 NC_003028.3|:1746104-1746280 SP1865 AAK75937.1 NC_003028.3|: c1772987-1771923 SP0904AAK75029.1 NC_003028.3|: c858126-857311 SP0765 AAK74903.1 NC_003028.3|:724170-725207 SP1634 AAK75714.1 NC_003028.3|: 1534348-1535421 SP0418AAK74581.1 NC_003028.3|: 396692-396916 SP1923 AAK75991.1 NC_003028.3|:c1833311-1831896 SP1313 AAK75991.1 NC_003028.3|: c1833311-1831896 SP0775AAK74913.1 NC_003028.3|: 731798-732070 SP0314 AAK74491.1 NC_003028.3|:287483-290683 SP0912 AAK75037.1 NC_003028.3|: 864707-865465 SP0159AAK74341.1 NC_003028.3|: c157554-156292 SP0910 AAK75035.1 NC_003028.3|:863462-863734 SP2148 AAK76205.1 NC_003028.3|: 2062144-2063373 SP1412AAK75510.1 NC_003028.3|: c1332393-1331605 SP0372 AAK74539.1NC_003028.3|: 350268-350597 SP1304 AAK75407.1 NC_003028.3|:c1232491-1232390 SP2002 AAK76069.1 NC_003028.3|: c1906183-1905446 SP0612AAK74764.1 NC_003028.3|: 579708-579806 SP1988 AAK76055.1 NC_003028.3|:c1892598-1890565 SP0484 AAK74643.1 NC_003028.3|: 465572-466402 SP0847AAK74978.1 NC_003028.3|: 794144-795202 SP1527 AAK75616.1 NC_003028.3|:c1439494-1437536 SP0542 AAK74699.1 NC_003028.3|: 515940-516059 SP0441AAK74602.1 NC_003028.3|: 414869-415057 SP0350 AAK74523.1 NC_003028.3|:323990-324625 SP0014 AAK74207.1 NC_003028.3|: 14450-14929 SP1965AAK76032.1 NC_003028.3|: c1873279-1873073 SP0117 AAK74303.1NC_003028.3|: 118423-120657 SP0981 AAK75102.1 NC_003028.3|:927115-928056 SP2229 AAK76277.1 NC_003028.3|: c2148627-2147602 SP2136AAK76194.1 NC_003028.3|: c2048521-2046656 SP1179 AAK75288.1NC_003028.3|: 1116230-1118389 SP1174 AAK75283.1 NC_003028.3|:c1110717-1108258 SP2216 AAK76264.1 NC_003028.3|: c2136445-2135267 SP1393AAK75491.1 NC_003028.3|: 1316756-1318027 SP1384 AAK75482.1 NC_003028.3|:c1309464-1308967 SP2032 AAK76097.1 NC_003028.3|: c1939994-1938321

Typically, the polypeptides present in compounds of the invention areimmunogenic, either alone or as a variant, which includes polypeptidesfused to another polypeptide or mixed with or complexed to an adjuvant.Variants also include sequences with less than 100% sequence identity,as described herein. In certain embodiments, an antigen of Table 1 or 2is provided as a full length polypeptide. In addition, one may usefragments, precursors and analogs that have an appropriateimmunogenicity.

These polypeptides may be immunogenic in mammals, for example mice,guinea pigs, or humans. An immunogenic polypeptide is typically onecapable of raising a significant immune response in an assay or in asubject. The immune response may be innate, humoral, cell-mediated, ormucosal (combining elements of innate, humoral and cell-mediatedimmunity). For instance, an immunogenic polypeptide may increase theamount of IL-17 produced by T cells. The IL-17 assay described inExamples 1-4 is an example of an assay that may be used to identify animmunogenic polypeptide. Alternatively or additionally, an immunogenicpolypeptide may (i) induce production of antibodies, e.g., neutralizingantibodies, that bind to the polypeptide and/or the whole bacteria, (ii)induce T_(H)17 immunity, (iii) activate the CD4⁺ T cell response, forexample by increasing CD4⁺ T cells and/or increasing localization ofCD4⁺ T cells to the site of infection or reinfection, (iv) activate theCD8⁺ CTL response, for example by increasing CD8⁺ T cells and/orincreasing localization of CD8⁺ T cells to the site of infection orreinfection, (v) induce T_(H)1 immunity, and/or (vi) activate innateimmunity. In some embodiments, an immunogenic polypeptide causes theproduction of a detectable amount of antibody specific to that antigen.

In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%,60% or 70% identity to human autoantigens and/or gut commensal bacteria(e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium,Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium,Escherichia and Lactobacillus species). Examples of human autoantigensinclude insulin, proliferating cell nuclear antigen, cytochrome P450,and myelin basic protein.

The present invention also provides an immunogenic compositioncomprising a pharmaceutically acceptable carrier, a polypeptide havingan amino acid sequence comprising SEQ ID NO: 265, 266, or 268 or animmunogenic fragment thereof, and one or more polypeptides having aminoacid sequences comprising any of SEQ ID NOS: 1-23 and SP1574, SP1655,SP2106, SP1473, SP0605, SP1177, SP0335, SP0906, SP1828, SP2157, SP1229,SP1128, SP1836, SP1865, SP0904, SP0765, SP1634, SP0418, SP1923, SP1313,SP0775, SP0314, SP0912, SP0159, SP0910, SP2148, SP1412, SP0372, SP1304,SP2002, SP0612, SP1988, SP0484, SP0847, SP1527, SP0542, SP0441, SP0350,SP0014, SP1965, SP0117, SP0981, SP2229, SP2136, SP1179, SP1174, SP2216,SP1393, SP0641.1, SP1384, and SP2032, or an immunogenic fragmentthereof.

In some embodiments, the vaccine formulation comprises at least twopolypeptides, each polypeptide belonging to a different group of(i)-(vii): (i) SEQ ID NO: 1 or an immunogenic fragment thereof, (ii) oneof SEQ ID NOS: 2-5 and 14-17 or an immunogenic fragment thereof, (iii)one of SEQ ID NOS: 6-7 and 18-19 or an immunogenic fragment thereof,(iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ IDNOS: 9-10 and 20-21 or an immunogenic fragment thereof, (vi) one of SEQID NOS: 11-13, 267, and 269-270 or an immunogenic fragment thereof, and(vii) one of SEQ ID NOS: 265-266 and 268 or an immunogenic fragmentthereof. Examples of such combinations are listed below. Additionalcombinations may be made by replacing one of the sequences below withthe corresponding consensus sequence, e.g., one of SEQ ID NOS: 14-21 or268-270. In some embodiments, one of the polypeptides is one of SEQ IDNOS: 265-266 and 268 or an immunogenic fragment thereof. In someembodiments, the vaccine formulation further comprises a pneumolysoid.In some embodiments, the vaccine formulation further comprises CbpA or aderivative thereof. In some embodiments, the vaccine formulation furthercomprises PspA or a derivative thereof comprising all or a fragment ofthe proline-rich region of PspA.

SEQ ID NO: 1 and SEQ ID NO: 2

SEQ ID NO: 1 and SEQ ID NO: 3

SEQ ID NO: 1 and SEQ ID NO: 4

SEQ ID NO: 1 and SEQ ID NO: 5

SEQ ID NO: 1 and SEQ ID NO: 6

SEQ ID NO: 1 and SEQ ID NO: 7

SEQ ID NO: 1 and SEQ ID NO: 8

SEQ ID NO: 1 and SEQ ID NO: 9

SEQ ID NO: 1 and SEQ ID NO: 10

SEQ ID NO: 1 and SEQ ID NO: 11

SEQ ID NO: 1 and SEQ ID NO: 12

SEQ ID NO: 1 and SEQ ID NO: 13

SEQ ID NO: 1 and SEQ ID NO: 265

SEQ ID NO: 1 and SEQ ID NO: 266

SEQ ID NO: 1 and SEQ ID NO: 267

SEQ ID NO: 2 and SEQ ID NO: 6

SEQ ID NO: 2 and SEQ ID NO: 7

SEQ ID NO: 2 and SEQ ID NO: 8

SEQ ID NO: 2 and SEQ ID NO: 9

SEQ ID NO: 2 and SEQ ID NO: 10

SEQ ID NO: 2 and SEQ ID NO: 11

SEQ ID NO: 2 and SEQ ID NO: 12

SEQ ID NO: 2 and SEQ ID NO: 13

SEQ ID NO: 2 and SEQ ID NO: 265

SEQ ID NO: 2 and SEQ ID NO: 266

SEQ ID NO: 2 and SEQ ID NO: 267

SEQ ID NO: 3 and SEQ ID NO: 6

SEQ ID NO: 3 and SEQ ID NO: 7

SEQ ID NO: 3 and SEQ ID NO: 8

SEQ ID NO: 3 and SEQ ID NO: 9

SEQ ID NO: 3 and SEQ ID NO: 10

SEQ ID NO: 3 and SEQ ID NO: 11

SEQ ID NO: 3 and SEQ ID NO: 12

SEQ ID NO: 3 and SEQ ID NO: 13

SEQ ID NO: 3 and SEQ ID NO: 265

SEQ ID NO: 3 and SEQ ID NO: 266

SEQ ID NO: 3 and SEQ ID NO: 267

SEQ ID NO: 4 and SEQ ID NO: 6

SEQ ID NO: 4 and SEQ ID NO: 7

SEQ ID NO: 4 and SEQ ID NO: 8

SEQ ID NO: 4 and SEQ ID NO: 9

SEQ ID NO: 4 and SEQ ID NO: 10

SEQ ID NO: 4 and SEQ ID NO: 11

SEQ ID NO: 4 and SEQ ID NO: 12

SEQ ID NO: 4 and SEQ ID NO: 13

SEQ ID NO: 4 and SEQ ID NO: 265

SEQ ID NO: 4 and SEQ ID NO: 266

SEQ ID NO: 4 and SEQ ID NO: 267

SEQ ID NO: 5 and SEQ ID NO: 6

SEQ ID NO: 5 and SEQ ID NO: 7

SEQ ID NO: 5 and SEQ ID NO: 8

SEQ ID NO: 5 and SEQ ID NO: 9

SEQ ID NO: 5 and SEQ ID NO: 10

SEQ ID NO: 5 and SEQ ID NO: 11

SEQ ID NO: 5 and SEQ ID NO: 12

SEQ ID NO: 5 and SEQ ID NO: 13

SEQ ID NO: 5 and SEQ ID NO: 265

SEQ ID NO: 5 and SEQ ID NO: 266

SEQ ID NO: 5 and SEQ ID NO: 267

SEQ ID NO: 6 and SEQ ID NO: 8

SEQ ID NO: 6 and SEQ ID NO: 9

SEQ ID NO: 6 and SEQ ID NO: 10

SEQ ID NO: 6 and SEQ ID NO: 11

SEQ ID NO: 6 and SEQ ID NO: 12

SEQ ID NO: 6 and SEQ ID NO: 13

SEQ ID NO: 6 and SEQ ID NO: 265

SEQ ID NO: 6 and SEQ ID NO: 266

SEQ ID NO: 6 and SEQ ID NO: 267

SEQ ID NO: 7 and SEQ ID NO: 8

SEQ ID NO: 7 and SEQ ID NO: 9

SEQ ID NO: 7 and SEQ ID NO: 10

SEQ ID NO: 7 and SEQ ID NO: 11

SEQ ID NO: 7 and SEQ ID NO: 12

SEQ ID NO: 7 and SEQ ID NO: 13

SEQ ID NO: 7 and SEQ ID NO: 265

SEQ ID NO: 7 and SEQ ID NO: 266

SEQ ID NO: 7 and SEQ ID NO: 267

SEQ ID NO: 8 and SEQ ID NO: 9

SEQ ID NO: 8 and SEQ ID NO: 10

SEQ ID NO: 8 and SEQ ID NO: 11

SEQ ID NO: 8 and SEQ ID NO: 12

SEQ ID NO: 8 and SEQ ID NO: 13

SEQ ID NO: 8 and SEQ ID NO: 265

SEQ ID NO: 8 and SEQ ID NO: 266

SEQ ID NO: 8 and SEQ ID NO: 267

SEQ ID NO: 9 and SEQ ID NO: 11

SEQ ID NO: 9 and SEQ ID NO: 12

SEQ ID NO: 9 and SEQ ID NO: 13

SEQ ID NO: 9 and SEQ ID NO: 265

SEQ ID NO: 9 and SEQ ID NO: 266

SEQ ID NO: 9 and SEQ ID NO: 267

SEQ ID NO: 10 and SEQ ID NO: 11

SEQ ID NO: 10 and SEQ ID NO: 12

SEQ ID NO: 10 and SEQ ID NO: 13

SEQ ID NO: 10 and SEQ ID NO: 265

SEQ ID NO: 10 and SEQ ID NO: 266

SEQ ID NO: 10 and SEQ ID NO: 267

SEQ ID NO: 11 and SEQ ID NO: 265

SEQ ID NO: 11 and SEQ ID NO: 266

SEQ ID NO: 12 and SEQ ID NO: 265

SEQ ID NO: 12 and SEQ ID NO: 266

SEQ ID NO: 13 and SEQ ID NO: 265

SEQ ID NO: 13 and SEQ ID NO: 266

In certain embodiments, the vaccine formulation comprises at least threedifferent polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 1-13, 265, 266, and 267, or an immunogenic fragment thereof,each polypeptide belonging to a different group of (i)-(vii): (i) SEQ IDNO: 1 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 2-5 oran immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 or animmunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenicfragment thereof, (v) one of SEQ ID NOS: 9-10 or an immunogenic fragmentthereof, (vi) one of SEQ ID NO: 11-13 and 267, or an immunogenicfragment thereof, and (vii) one of SEQ ID NOS: 265-266 or an immunogenicfragment thereof. Examples of such combinations are listed below.Additional combinations may be made by replacing one of the sequencesbelow with the corresponding consensus sequence, e.g., one of SEQ IDNOS: 14-21 or 268-270. In some embodiments, one of the polypeptides isone of SEQ ID NOS: 265-266 and 268 or an immunogenic fragment thereof.In some embodiments, the vaccine formulation further comprises apneumolysoid. In some embodiments, the vaccine formulation furthercomprises CbpA or a derivative thereof. In some embodiments, the vaccineformulation further comprises PspA or a derivative thereof comprisingall or a fragment of the proline-rich region of PspA.

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 1, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 1, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 267

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 267

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 2, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 2, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 2, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 267

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 267

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 3, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 3, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 3, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 267

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 267

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 4, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 4, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 4, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 267

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 267

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 5, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 5, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 6, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 6, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 267

SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 7, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 7, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 266

SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 267

SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 8, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 265

SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 266

SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 267

SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 9, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 9, SEQ ID NO: 267, and SEQ ID NO: 266

SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 265

SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 266

SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 265

SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 266

SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 265

SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 266

SEQ ID NO: 10, SEQ ID NO: 267, and SEQ ID NO: 265

SEQ ID NO: 10, SEQ ID NO: 267, and SEQ ID NO: 266

In some embodiments, the vaccine formulation comprises at least twodifferent polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 14-21, 268, 269 and 270, or an immunogenic fragment thereof.In certain such embodiments, the vaccine formulation comprises at leasttwo polypeptides, each polypeptide belonging to a different group of(i)-(v): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragmentthereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragmentthereof; (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragmentthereof, (iv) one of SEQ ID NO: 268 or an immunogenic fragment thereof,and (v) one of SEQ ID NOS: 269-279 or an immunogenic fragment thereof.Examples of such combinations are listed below. The combinations belowspecify consensus sequences. However, additional combinations may bemade by replacing one of the consensus sequences with the correspondingnon-consensus sequence, e.g., one of SEQ ID NOS: 1-13 or 266-267. Insome embodiments, one of the polypeptides is SEQ ID NO: 268 or animmunogenic fragment thereof. In some embodiments, the vaccineformulation further comprises a pneumolysoid. In some embodiments, thevaccine formulation further comprises CbpA or a derivative thereof. Insome embodiments, the vaccine formulation further comprises PspA or aderivative thereof comprising all or a fragment of the proline-richregion of PspA.

SEQ ID NO: 14 and SEQ ID NO: 18

SEQ ID NO: 14 and SEQ ID NO: 19

SEQ ID NO: 14 and SEQ ID NO: 20

SEQ ID NO: 14 and SEQ ID NO: 21

SEQ ID NO: 14 and SEQ ID NO: 268

SEQ ID NO: 14 and SEQ ID NO: 269

SEQ ID NO: 14 and SEQ ID NO: 270

SEQ ID NO: 15 and SEQ ID NO: 18

SEQ ID NO: 15 and SEQ ID NO: 19

SEQ ID NO: 15 and SEQ ID NO: 20

SEQ ID NO: 15 and SEQ ID NO: 21

SEQ ID NO: 15 and SEQ ID NO: 268

SEQ ID NO: 15 and SEQ ID NO: 269

SEQ ID NO: 15 and SEQ ID NO: 270

SEQ ID NO: 16 and SEQ ID NO: 18

SEQ ID NO: 16 and SEQ ID NO: 19

SEQ ID NO: 16 and SEQ ID NO: 20

SEQ ID NO: 16 and SEQ ID NO: 21

SEQ ID NO: 16 and SEQ ID NO: 268

SEQ ID NO: 16 and SEQ ID NO: 269

SEQ ID NO: 16 and SEQ ID NO: 270

SEQ ID NO: 17 and SEQ ID NO: 18

SEQ ID NO: 17 and SEQ ID NO: 19

SEQ ID NO: 17 and SEQ ID NO: 20

SEQ ID NO: 17 and SEQ ID NO: 21

SEQ ID NO: 17 and SEQ ID NO: 268

SEQ ID NO: 17 and SEQ ID NO: 269

SEQ ID NO: 17 and SEQ ID NO: 270

SEQ ID NO: 18 and SEQ ID NO: 20

SEQ ID NO: 18 and SEQ ID NO: 21

SEQ ID NO: 18 and SEQ ID NO: 268

SEQ ID NO: 18 and SEQ ID NO: 269

SEQ ID NO: 18 and SEQ ID NO: 270

SEQ ID NO: 19 and SEQ ID NO: 20

SEQ ID NO: 19 and SEQ ID NO: 21

SEQ ID NO: 19 and SEQ ID NO: 268

SEQ ID NO: 19 and SEQ ID NO: 269

SEQ ID NO: 19 and SEQ ID NO: 270

SEQ ID NO: 20 and SEQ ID NO: 268

SEQ ID NO: 20 and SEQ ID NO: 269

SEQ ID NO: 20 and SEQ ID NO: 270

SEQ ID NO: 21 and SEQ ID NO: 268

SEQ ID NO: 21 and SEQ ID NO: 269

SEQ ID NO: 21 and SEQ ID NO: 270

SEQ ID NO: 268 and SEQ ID NO: 269

SEQ ID NO: 268 and SEQ ID NO: 270

In some embodiments, the fragment is a truncated fragment of any of SEQID NOS: 14-21 and 268-270 wherein from 1-20 amino acid residues areremoved from the N-terminus, C-terminus, or both.

In some embodiments, the vaccine formulation comprises a polypeptidehaving an amino acid sequence comprising any of SEQ ID NOS: 14-17. Insome embodiments, the vaccine formulation comprises a polypeptide havingan amino acid sequence comprising either of SEQ ID NOS: 18-19. In someembodiments, the vaccine formulation comprises a polypeptide having anamino acid sequence comprising either of SEQ ID NOS: 20-21. In someembodiments, the vaccine formulation comprises a polypeptide having anamino acid sequence comprising any of SEQ ID NOS: 268-270.

In some aspects, a vaccine formulation comprising one or more of SEQ IDNOS: 14-21, 268, 269 and 270 further comprises a polypeptide having anamino acid sequence comprising any of SEQ ID NOS: 1-13, 265, 266 and267.

In certain embodiments, the vaccine formulation comprises at least threedifferent polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 14-21, 268, 269 and 270, or an immunogenic fragment thereof.In certain such embodiments, the vaccine formulation comprises three of(i)-(v): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragmentthereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragmentthereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragmentthereof, (iv) one of SEQ ID NO: 268 or an immunogenic fragment thereof,and (v) one of SEQ ID NOS: 269-270 or an immunogenic fragment thereof.Examples of such combinations are listed below. The combinations belowspecify consensus sequences. However, additional combinations may bemade by replacing one of the consensus sequences with the correspondingnon-consensus sequence, e.g., one of SEQ ID NOS: 1-13 or 266-267. Insome embodiments, one of the polypeptides is SEQ ID NO: 268 or animmunogenic fragment thereof. In some embodiments, the vaccineformulation further comprises a pneumolysoid. In some embodiments, thevaccine formulation further comprises CbpA or a derivative thereof. Insome embodiments, the vaccine formulation further comprises PspA or aderivative thereof comprising all or a fragment of the proline-richregion of PspA.

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 268

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 269

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 270

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 268

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 269

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 270

SEQ ID NO: 14, SEQ ID NO: 268, and SEQ ID NO: 269

SEQ ID NO: 14, SEQ ID NO: 268, and SEQ ID NO: 270

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 268

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 269

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 270

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 268

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 269

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 270

SEQ ID NO: 15, SEQ ID NO: 268, and SEQ ID NO: 269

SEQ ID NO: 15, SEQ ID NO: 268, and SEQ ID NO: 270

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 268

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 269

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 270

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 268

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 269

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 270

SEQ ID NO: 16, SEQ ID NO: 268, and SEQ ID NO: 269

SEQ ID NO: 16, SEQ ID NO: 268, and SEQ ID NO: 270

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 268

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 269

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 270

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 268

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 269

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 270

SEQ ID NO: 17, SEQ ID NO: 268, and SEQ ID NO: 269

SEQ ID NO: 17, SEQ ID NO: 268, and SEQ ID NO: 270

SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 268

SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 269

SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 270

SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 268

SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 269

SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 270

SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 268

SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 269

SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 270

SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 268

SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 269

SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 270

SEQ ID NO: 20, SEQ ID NO: 268, and SEQ ID NO: 269

SEQ ID NO: 20, SEQ ID NO: 268, and SEQ ID NO: 270

SEQ ID NO: 21, SEQ ID NO: 268, and SEQ ID NO: 269

SEQ ID NO: 21, SEQ ID NO: 268, and SEQ ID NO: 270

A polypeptide may comprise one or more immunogenic portions and one ormore non-immunogenic portions. The immunogenic portions may beidentified by various methods, including protein microarrays,ELISPOT/ELISA techniques, and/or specific assays on different deletionmutants (e.g., fragments) of the polypeptide in question. Immunogenicportions may also be identified by computer algorithms. Some suchalgorithms, like EpiMatrix (produced by EpiVax), use a computationalmatrix approach. Other computational tools for identifying antigenicepitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted byDana Farber Cancer Institute on the world wide web atimmunax.dfci.harvard.edu/PEPVAC), MHCPred (which uses a partial leastsquares approach and is hosted by The Jenner Institute on the world wideweb at www.jenner.ac.uk/MHCPred), and Immune Epitope Database algorithmson the world wide web at tools.immuneepitope.org. An immunogenicfragment of a polypeptide described herein comprises at least oneimmunogenic portion, as measured experimentally or identified byalgorithm. Peptides identified by the tools described above include thefollowing:

SP0148 SP1634 SP0882 SP0314 SP2108 Fragments Fragments FragmentsFragments Fragments (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS(SEQ ID NOS 34- 58-82, 83-109, 110-130, 131-169, 57, respectively, inrespectively, in respectively, respectively, in respectively, inorder of order of in order of order of order of appearance) appearance)appearance) appearance) appearance) AIIDGPWKA ALGLVAAGV RLLDLAPQVHLDNLVLKV MLKDKIAFL VMMAPYDRV ELTGYEIEV MLEIPAHQI DLIAGRVHL SLADYTYKVSIAGINYAK AVNNLSYTK KNFFAHHPK ILLPKDYEK FLLLGAFYL VWDPAKNML TYLPAEADIKVILAGHSK EYQDQIGCL VLIDGLSQL QPLPNISQM RYNMAVNNL SFDNLVSTL YFHDGQNVFILASLGFLL APYDRVGSL DFQQIMVRL YYDLPLNEL NPDISRMIV GLSQLLPVI APAVIESLVEHTDNPTIL YFDLFFGTI IPWSENLPD FLLNHYMTV FYYTYGLLA APIAQNPNV ALEYIHHLFQFGGKGVEY MLIPNVDRA SKYAFAGE LPSDQQPYV LPLNELDIL IGLEYQDQI KLEEMAKQVTEGAGNLI YVYPLLAQG IPQGSIIGM VYFHDGQN VLKRGVYTI LADWTNFYY QGLDNLKVIDPELQKQFA MEVVKPFI KVIAGLLRK SLVMYYNKD KYLYAAPI AVYTFDAPG YLKMKEHKLTLNYEHMNK KEAGVKVTL GELTGYEI QSLTPEERE KLSPDQRIF NIGYFFFKK KSTAVLGTVNPNVLVVKK AIYAASQI RIFIYVGTE KYTDVIEKF GAKTDDTTK KLSKQFFGD LEIPAHQIFIDETYRTK KYDDSVSTI SQKFVDFLV GSPRPFIYE LLDLAPQVP DTDRSYPVV TFNQMIKELQAFKDAKVN AVNNLSYTK WQIEDKHFV YIDSSLCYY DYPETQSVF AVIESLVMY KIFDKIGVETLGRLTQLL TQFIGLEYQ TPRAINNTL DAKTAANDA MVRLSDGQF LYFDLFFGT KDTDRSYPVAPLLVNGEL YGVATIPTL YVYPLLAQG SINDLASLK LCYYHDLIA YIDHTNVAY KTAAIIDGPVVQATTSAK SINDLASLK NVFNSKESF KQNGDSYGY KAYEKEAGV TLEKLSKQF YYDLPLNELFLLNHYMTV AGNGAYVFG VAAGVLAAC QKVILAGHS FYLYNGDLS AWVIPQAVK LDNLKVIELGTDDSIIGW KSFAPLLV NMAVNNLSY TYLSFDNLV DETVVRTV FGTILDAGI YIDHTNVAYNQITAVYTF MLKDKIAFL KLRFKIKTD KLELFYETG KIAFLGSNI SVPRTSYLS FGFGLSLFSSTIRSIEQV FRKTTDNPF TVVRTVRDS STIRSIEQV DGLSQLLPV FGFGLSLFS KLVDQGEGF

SP0024 Fragments (SEQ ID NOS 170- SP1072 Fragments SP0641 FragmentsSP1912 Fragments 193, respectively, (SEQ ID NOS 194- (SEQ ID NOS 228-(SEQ ID NOS 277-300, in order of 227, respectively, in264, respectively, in respectively, in order of appearance)order of appearance) order of appearance) appearance) AIVTCMDSRGIEVEKPLY AAYAPNEVV KMWMAGLALLGIGSL AQTFENEPF AEAHLLYRM AGDLRGKIILLGIGSLALATKKVA AYVALHGQL ALLNQDNMR DEIANEVWY MAGLALLGIGSLALA DDVIISGAIAPPERNYLY DNYLIYGDL WMAGLALLGIGSLAL FENEPFQEY AQNSYIHIL DQKEHPEKFGLALLGIGSLALATK FMQANQAYV AVASMGTAL DSLTDRLKL LALLGIGSLALATKK ISQQQMGTRAYLLTKTRI EAKNKNKFV FSDMGEIATLYVQVY KPKTRVAIV DAAKFYHAI EGQGRNRKLKAKKMWMAGLALLGI LHGQLNLPL DTALEELER EIKGAGDLR ALLGIGSLALATKKVAKLHVAQALGL EEYQGVPFI EPIAEGQYF KMWMAGLALLGIG LPLKPKTRV EFLEKIAPLEVSELKPHR SDMGEIATLYVQVYE MGTREIVVL EFQVLYDLL GAFFDKSKI DMGEIATLYVQVYESMQLLIESPL EHVEHLKRL GDLKWDGLI AGLALLGIGSLALAT QANQAYVAL ELSEVEMTRGEVEKNLEV MGEIATLYVQVYESS QFMQANQAY ESPLVLNDY IHFESVEEM KKMWMAGLALLGIGSQLNLPLKPK GEKTPSFNV IMFIVGIFL GMKAKKMWMAGLALL QQMGTREIV GLCPFHGEKIPGTLNKGI MKAKKMWMAGLALLG REIVVLHHT IGDMPVQIV IRYQVFTFK HFSDMGEIATLYVQVSPLIPDDVI ITMPVTKQL ISDKGGFNW MNGMKAKKMWMAGLA SRLHVAQAL KALLNQDNMIVSEEDFIL MWMAGLALLGIGSLA TEDMIRSLV KRLTKKLVL KEIGVEEAI DHFSDMGEIATLYVQVDVSDQDFL LTKTRISPI KIVVKDFAR RDHFSDMGEIATLYV VSDQDFLPF LVLVYDGDKKKINFQPSL NGMKAKKMWMAGLAL VTEDMIRSL MRAEAHLLY KLKFVYIGK NGPEDLAYLKVYYGNNYK QTEEVERAW KYWQAIRAL SEIYLMEGF LHIDNTRDF SPHQALYDM MRFKKEDLKVDKQVIEEI NESVVDNYL VEMTRNKAL NEVWYAGAA VLYDLLGQY NINDIVDGL VPFIEAVQIQYLLKDNII WYQVLAQDL SPRQQGAGL YLMEGFMDV SRSKTLGGY SSLKNTKVL TAAVILAAYWTELPAMGY

Thus, in some aspects, this application provides an immunogenic fragmentof an antigen described herein. The fragments, in some instances, areclose in size to the full-length polypeptide or the polypeptide of Table1 or 2. For example, they may lack at most one, two, three, four, five,ten, twenty, or thirty amino acids from one or both termini. In certainembodiments, the polypeptide is 100-500 amino acids in length, or150-450, or 200-400, or 250-250 amino acids in length. In someembodiments, the polypeptide is 100-200, 150-250, 200-300, 250-350,300-400, 350-450, or 400-500 amino acids in length. In certainembodiments, the fragments result from processing, or partialprocessing, of signal sequences by an expression host, e.g. E. coli, aninsect cell line (e.g., the baculovirus expression system), or amammalian (e.g., human or Chinese Hamster Ovary) cell line. Thefragments described above or sub-fragments thereof (e.g., fragments of8-50, 8-30, or 8-20 amino acid residues) preferably have one of thebiological activities described below, such as increasing the amount ofIL-17 released by at least 1.5 fold or 2 fold or more (e.g., either asan absolute measure or relative to an immunologically inactive protein).A fragment may be used as the polypeptide in the vaccines describedherein or may be fused to another protein, protein fragment or apolypeptide.

In some embodiments, the fragment is a truncated fragment of any of SEQID NOS: 1-21 or 265-270, having from 1-5, 1-10, or 1-20 amino acidresidues removed from the N-terminus, C-terminus, or both. In some suchembodiments, the same number of residues is removed from the N-terminusand the C-terminus, while in other embodiments, a different number ofresidues is removed from the N-terminus compared to the C-terminus.

In certain aspects, this application provides immunogenic polypeptideswith at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to apolypeptide of Table 1 or 2. In certain embodiments, the vaccineformulation comprises at least two different polypeptides having anamino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99%identical to any of SEQ ID NOS: 1-21 or 265-270, or an immunogenicfragment thereof.

In some embodiments, one or more, e.g., two, three, four, or morepolypeptides from Table 1 or 2 or immunogenic fragments or variantsthereof are provided in a mixture. In some embodiments, the mixturecontains both full-length polypeptides and fragments resulting fromprocessing, or partial processing, of signal sequences by an expressionhost, e.g. E. coli, an insect cell line (e.g., the baculovirusexpression system), or a mammalian (e.g., human or Chinese HamsterOvary) cell line.

In some embodiments, rather than being in a simple physical mixture,two, three, four, or more polypeptides from Table 1 or 2 or immunogenicfragments or variants thereof are covalently bound to each other, e.g.as a fusion protein. In some embodiments, the vaccine formulationcontains substantially no other S. pneumoniae polypeptides other thanpolypeptides having an amino acid sequence comprising any of SEQ ID NOS:1-23 or 265-270. In some embodiments, the vaccine formulation containssubstantially no other S. pneumoniae polypeptides other thanpolypeptides of Table 1. In some embodiments, the vaccine formulationcontains substantially no other S. pneumoniae polypeptides other thanpolypeptides of Tables 1 and/or 2.

In certain embodiments, vaccine formulations or immunogenic compositionscontain substantially no other S. pneumoniae polypeptides other thanpolypeptides having an amino acid sequence comprising any of SEQ ID NO:1-23 or 265-270. In certain such embodiments, vaccine formulations orimmunogenic compositions contain substantially no other S. pneumoniaepolypeptides other than polypeptides having an amino acid sequenceconsisting of any of SEQ ID NO: 1-23 or 265-270. In some embodiments,vaccine formulations or immunogenic compositions contain substantiallyno other S. pneumoniae polypeptides other than polypeptides having anamino acid sequence comprising (or consisting of) any of the amino acidsequences of the polypeptides of Tables 1 and/or 2. Substantially, inthis context, refers to less than 50%, less than 40%, less than 30%,less than 20%, less than 10%, less than 5%, less than 3%, less than 2,or even less than 1% of the other S. pneumoniae polypeptides.

In certain embodiments, the vaccine composition induces a T_(H)17 cellresponse at least 1.5-fold greater than that induced by a controlunrelated antigen (such as the HSV-2 protein ICP47 with the gene nameUS12) after contacting T_(H)17 cells. In some embodiments, the vaccineformulation inhibits infection by S. pneumoniae in an uninfectedsubject. In certain embodiments, the vaccine formulation reducesoccurrence or duration of S. pneumoniae nasopharyngeal colonization inan individual infected by S. pneumoniae. In some embodiments, thevaccine formulation inhibits development of sepsis in an individualinfected by S. pneumoniae. In some embodiments, the vaccine formulationinhibits development of pneumonia, meningitis, otitis media, sinusitisor infection of other sites or organs with S. pneumoniae.

In certain embodiments, this application provides nucleic acids encodingone or more of the polypeptides described above, such as DNA, RNA, or ananalog thereof. The underlying DNA sequences for the polypeptidesdescribed above may be modified in ways that do not affect the sequenceof the protein product, and such sequences are included in theinvention. For instance, the DNA sequence may be codon-optimized toimprove expression in a host such as E. coli, an insect cell line (e.g.,using the baculovirus expression system), or a mammalian (e.g., human orChinese Hamster Ovary) cell line.

In certain embodiments, this application provides nucleic acids (such asDNA, RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%,97%, 98%, 99%, or 100% identical to a gene in Table 1 or 2, or a variantor portion of said gene. In certain embodiments, the nucleic acid is600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In someembodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000nucleotides in length. The nucleic acids may be used, for example, forrecombinant production of the polypeptides of Tables 1 and 2, orimmunogenic fragments thereof.

In some embodiments, the vaccine or immunogenic composition may comprisefusion proteins and/or fusion DNA constructs. The polypeptides describedherein may be used without modification. In certain embodiments, whensmaller related polypeptides are used, such as fragments or the like,and their molecular weight is less than about 5000 daltons, e.g., 1500to 5000 daltons, modification may be useful in eliciting the desiredimmune response. For example, the smaller polypeptides can be conjugatedto an appropriate immunogenic carrier such as tetanus toxoid,pneumolysin, keyhole limpet hemocyanin or the like.

In certain embodiments, the vaccine formulation comprises at least onelipidated polypeptide. Conjugation to the lipid moiety may be direct orindirect (e.g., via a linker). The lipid moiety may be synthetic ornaturally produced. In certain embodiments, a polypeptide from Table 1or 2 may be chemically conjugated to a lipid moiety. In certainembodiments, a construct may comprise a gene or polypeptide from Table 1or 2, or an immunogenic fragment or variant thereof, and a lipidationsequence including a lipobox motif. A canonical lipobox motif is shownas SEQ ID NO: 274. A lipidation sequence may be N-terminal or C-terminalto the protein, and may be embedded in a signal or other sequence, or ina fusion protein. Exemplary lipidation sequences include the signalsequence of SP2108 (SEQ ID NO: 275) and the signal sequence of the E.coli gene RlpB (SEQ ID NO: 276). A signal sequence may be, for example,an E. coli or S. pneumoniae signal sequence. Exemplary E. coli signalsequences include the mlpA signal sequence (Lin, J. J. et al., “AnEscherichia coli mutant with an amino acid alteration within the signalsequence of outer membrane prolipoprotein” Proc Natl Acad Sci USA. 1978October; 75 (10):4891-5), the lamB signal sequence (Emr, S. D. et al.“Mutations altering the cellular localization of the phage lambdareceptor, an Escherichia coli outer membrane protein”, Proc Natl AcadSci USA. 1978 December; 75 (12):5802-6), the MBP signal sequence(Bassford, P. J., “Use of gene fusion to study secretion ofmaltose-binding protein into Escherichia coli periplasm” J. Bacteriol.1979 July; 139 (1):19-31). Lpp is an exemplary E. coli signal sequencethat directs lipidation (Cullen, P. A. et al. “Construction andevaluation of a plasmid vector for the expression of recombinantlipoproteins in Escherichia coli” Plasmid. 2003 January; 49 (1):18-29.)E. coli signal sequences that direct lipidation are also described inLegrain, M. et al. (“Production of lipidated meningococcal transferrinbinding protein 2 in Escherichia coli” Protein Expr Purif. 1995 October;6 (5):570-8), e.g. the signal sequence of the gene RlpB (SEQ ID NO: 276)Numerous S. pneumoniae signal sequences are known in the art. One suchsignal sequence is SEQ ID NO: 275.

In other embodiments, a construct may comprise a gene or protein fromTable 1 or 2, or an immunogenic fragment or variant thereof, and a tag.A tag may be N-terminal or C-terminal. For instance, tags may be addedto the nucleic acid or polypeptide to facilitate purification,detection, solubility, or confer other desirable characteristics on theprotein or nucleic acid. For instance, a purification tag may be apeptide, oligopeptide, or polypeptide that may be used in affinitypurification. Examples include His, GST, TAP, FLAG, myc, HA, MBP, VSV-G,thioredoxin, V5, avidin, streptavidin, BCCP, Calmodulin, Nus, S tags,lipoprotein D, and β galactosidase. Particular exemplary His tagsinclude HHHHHH (SEQ ID NO: 32) and MSYYHHHHHH (SEQ ID NO: 33). In otherembodiments, the polypeptide is free of tags such as proteinpurification tags, and is purified by a method not relying on affinityfor a purification tag. In some embodiments, the fused portion is short.This, in some instances, the fusion protein comprises no more than 1, 2,3, 4, 5, 10, or 20 additional amino acids on one or both termini of thepolypeptide of Table 1 or 2.

B. Immunogenic Compositions

The present disclosure also provides pharmaceutical compositionscontaining immunogenic polypeptides or polynucleotides encoding theseimmunogenic polypeptides together with a pharmaceutical carrier.Antigens from S. pneumoniae were identified by screening immune cellsfrom mice infected with S. pneumoniae, or from healthy human donors. Thehuman donors had presumably been exposed to S. pneumoniae at some pointduring their lifetimes, because S. pneumoniae is a very common diseaseand colonizing pathogen. Briefly, a library of S. pneumoniae antigenswas expressed in bacteria and mixed with antigen presenting cells(APCs). The APCs, in turn, presented S. pneumoniae-derived polypeptidesto lymphocytes that had been isolated from mice or from human donors.Lymphocyte responses were assayed for reactivity to S. pneumoniae. Humandonors, as well as mice immunized with S. pneumoniae, producedlymphocytes specific to S. pneumoniae antigens. Thus, the presentdisclosure contemplates compositions of the S. pneumoniae antigens thatelicit a strong immune response in immunized or infected mice or humansfor counteracting infection by S. pneumoniae.

Tables 1 and 2 list the protein sequence and corresponding nucleotidesequence for S. pneumoniae antigens identified according to thescreening methods described herein. The antigens were identified inscreens of mouse and human T cells. In the screens of mouse T cells, theidentified antigens were subjected to at least two rounds of screening:a genome-wide round to identify pools of 4 antigens that elicited animmune response, followed by a deconvolution round to individually testand identify single antigens that elicited an immune response from apool identified in the genome-wide round. In contrast, in the screens ofhuman T cells, two different sets of antigen pools were created, suchthat a polypeptide was combined with different polypeptides between thefirst and second pools. Consequently, it is possible to determine whichpolypeptides are antigens by identifying which polypeptides are inpositive pools in both the first and second sets. Table 1 lists antigens(and variants thereof) that were identified by one of the abovescreening methods, and were subsequently subjected to further testing inthe mouse models described in Examples 5-12. Thus, compositionsaccording to this disclosure may include one or two or more of the geneslisted in Table 1 or 2, or the corresponding gene products.

An immunogenic composition may also comprise portions of saidStreptococcus polypeptides, for example deletion mutants, truncationmutants, oligonucleotides, and peptide fragments. In some embodiments,the portions of said polypeptides are immunogenic. The immunogenicity ofa portion of a protein is readily determined using the same assays thatare used to determine the immunogenicity of the full-length protein. Insome embodiments, the portion of the polypeptide has substantially thesame immunogenicity as the full-length proteins. In some embodiments,the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less thanthat of the full-length protein (e.g., polypeptides of Tables 1 and 2).The peptide fragments may be, for example, linear, circular, orbranched.

Some embodiments of the vaccine formulations and immunogeniccompositions described herein include an immunogenic polypeptide (e.g.,a polypeptide of Table 1 or 2) that contains a membrane translocatingsequence (MTS), to facilitate introduction of the polypeptide into themammalian cell and subsequent stimulation of the cell-mediated immuneresponse. Exemplary membrane translocating sequences include hydrophobicregion in the signal sequence of Kaposi fibroblast growth factor, theMTS of α-synuclein, β-synuclein, or γ-synuclein, the third helix of theAntennapedia homeodomain, SN50, integrin β3 h-region, HIV Tat, pAntp,PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and thoseMTSs described in U.S. Pat. Nos. 6,248,558, 6,432,680 and 6,248,558.

In certain embodiments, an antigen (e.g., a polypeptide of Table 1 or 2)is covalently bound to another molecule. This may, for example, increasethe half-life, solubility, bioavailability, or immunogenicity of theantigen. Molecules that may be covalently bound to the antigen include acarbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid,N-propionylated polysialic acid, nucleic acids, polysaccharides, andPLGA. There are many different types of PEG, ranging from molecularweights of below 300 g/mol to over 10,000,000 g/mol. PEG chains can belinear, branched, or with comb or star geometries. In some embodiments,the naturally produced form of a protein is covalently bound to a moeitythat stimulates the immune system. An example of such a moeity is alipid moeity. In some instances, lipid moieties are recognized by aToll-like receptor (TLR) such as TLR-2 or TLR-4, and activate the innateimmune system.

C. Antibodies Specific to the Proteins of Tables 1 and 2

Another aspect disclosed herein is an antibody preparation generatedagainst an antigenic composition (e.g., one of the proteins listed inTable 1 or 2 or an immunogenic fragment thereof). For instance, thisdisclosure provides combinations of two, three, four, or five antibodieseach recognizing a different protein of Table 1 or 2. Any of a varietyof antibodies are included. Such antibodies include, e.g., polyclonal,monoclonal, recombinant, humanized or partially humanized, single chain,Fab, and fragments thereof, etc. The antibodies can be of any isotype,e.g., IgG, various IgG isotypes such as IgG1, IgG2, IgG2a, IgG2b, IgG3,IgG4, etc.; and they can be from any animal species that producesantibodies, including goat, rabbit, mouse, chicken or the like. In someembodiments, Fab molecules are expressed and assembled in a geneticallytransformed host like E. coli. A lambda vector system is available thusto express a population of Fab's with a potential diversity equal to orexceeding that of subject generating the predecessor antibody. See Huseet al. (1989), Science 246, 1275-81.

D. Components of a Vaccine or Immunogenic Composition Comprising S.pneumoniae Antigens or Antibodies Recognizing the Same

In certain embodiments, the vaccine or immunogenic composition comprisesan antigen and one or more of the following: an adjuvant, stabilizer,buffer, surfactant, controlled release component, salt, preservative,and/or an antibody specific to said antigen.

1. Adjuvants

The vaccine formulations and immunogenic compositions described hereinmay include an adjuvant. Adjuvants can be broadly separated into twoclasses, based on their principal mechanisms of action: vaccine deliverysystems and immunostimulatory adjuvants (see, e.g., Singh et al., Curr.HIV Res. 1:309-20, 2003). In many vaccine formulations, the adjuvantprovides a signal to the immune system so that it generates a responseto the antigen, and the antigen is required for driving the specificityof the response to the pathogen. Vaccine delivery systems are oftenparticulate formulations, e.g., emulsions, microparticles,immune-stimulating complexes (ISCOMs), nanoparticles, which may be, forexample, particles and/or matrices, and liposomes. In contrast,immunostimulatory adjuvants are sometimes derived from pathogens and canrepresent pathogen associated molecular patterns (PAMP), e.g.,lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containingDNA, which activate cells of the innate immune system.

Alternatively, adjuvants may be classified as organic and inorganic.Inorganic adjuvants include alum salts such as aluminum phosphate,amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide,which are commonly used in human vaccines. Organic adjuvants compriseorganic molecules including macromolecules. An example of an organicadjuvant is cholera toxin.

Adjuvants may also be classified by the response they induce. In someembodiments, the adjuvant induces the activation of T_(H)1 cells orT_(H)2 cells. In other embodiments, the adjuvant induces the activationof B cells. In yet other embodiments, the adjuvant induces theactivation of antigen-presenting cells. These categories are notmutually exclusive; in some cases, an adjuvant activates more than onetype of cell.

In certain embodiments, the adjuvant induces the activation of T_(H)17cells. It may promote the CD4⁺ or CD8⁺ T cells to secrete IL-17. In someembodiments, an adjuvant that induces the activation of T_(H)17 cells isone that produces at least a 2-fold, and in some cases a 10-fold,experimental sample to control ratio in the following assay. In theassay, an experimenter compares the IL-17 levels secreted by twopopulations of cells: (1) cells from animals immunized with the adjuvantand a polypeptide known to induce T_(H)17 activation, and (2) cells fromanimals treated with the adjuvant and an irrelevant (control)polypeptide. An adjuvant that induces the activation of T_(H)17 cellsmay cause the cells of population (1) to produce more than 2-fold, ormore than 10-fold more IL-17 than the cells of population (2). IL-17 maybe measured, for example, by ELISA or ELISPOT. Certain toxins, such ascholera toxin and labile toxin (produced by enterotoxigenic E. coli, orETEC), activate a T_(H)17 response. Thus, in some embodiments, theadjuvant is a toxin. Cholera toxin was successfully used in the mousemodel to induce protective immunity in conjunction with certainpolypeptides from Table 1 (see Examples 5-8). One form of labile toxinis produced by Intercell. Mutant derivates of labile toxin that areactive as adjuvants but significantly less toxic can be used as well.Exemplary detoxified mutant derivatives of labile toxin include mutantslacking ADP-ribosyltransferase activity. Particular detoxified mutantderivatives of labile toxin include LTK7 (Douce et al., “Mutants ofEscherichia coli heat-labile toxin lacking ADP-ribosyltransferaseactivity act as nontoxic, mucosal adjuvants” PNAS Vol. 92, pp.1644-1648, February 1995) and LTK63 (Williams et al., “Innate Imprintingby the Modified Heat-Labile Toxin of Escherichia coli (LTK63) ProvidesGeneric Protection against Lung Infectious Disease” The Journal ofImmunology, 2004, 173: 7435-7443), LT-G192 (Douce et al. “Geneticallydetoxified mutants of heat-labile toxin from Escherichia coli are ableto act as oral adjuvants” Infect Immun. 1999 September; 67 (9):4400-6),and LTR72 (“Mucosal adjuvanticity and immunogenicity of LTR72, a novelmutant of Escherichia coli heat-labile enterotoxin with partial knockoutof ADP-ribosyltransferase activity.” J Exp Med. 1998 Apr. 6; 187(7):1123-32).

In some embodiments, the adjuvant comprises a VLP (virus-like particle).One such adjuvant platform, Alphavirus replicons, induces the activationof T_(H)17 cells using alphavirus and is produced by Alphavax. Incertain embodiments of the Alphavirus replicon system, alphavirus may beengineered to express an antigen of interest, a cytokine of interest(for example, IL-17 or a cytokine that stimulates IL-17 production), orboth, and may be produced in a helper cell line. More detailedinformation may be found in U.S. Pat. Nos. 5,643,576 and 6,783,939. Insome embodiments, a vaccine formulation is administered to a patient incombination with a nucleic acid encoding a cytokine.

Certain classes of adjuvants activate toll-like receptors (TLRs) inorder to activate a T_(H)17 response. TLRs are well known proteins thatmay be found on leukocyte membranes, and recognize foreign antigens(including microbial antigens). Administering a known TLR ligandtogether with an antigen of interest (for instance, as a fusion protein)can promote the development of an immune response specific to theantigen of interest. One exemplary adjuvant that activates TLRscomprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has beenproduced as a detoxified lipopolysaccharide (LPS) endotoxin obtainedfrom gram negative bacteria, such as S. minnesota. In particular,sequential acid and base hydrolysis of LPS produces an immunoactivelipid A fraction (which is MPL), and lacks the saccharide groups and allbut one of the phosphates present in LPS. A number of synthetic TLRagonists (in particular, TLR-4 agonists) are disclosed in Evans J T etal. “Enhancement of antigen-specific immunity via the TLR-4 ligands MPLadjuvant and Ribi.529.” Expert Rev Vaccines 2003 April; 2 (2):219-29.Like MPL adjuvants, these synthetic compounds activate the innate immunesystem via TLR. Another type of TLR agonist is a synthetic phospholipiddimer, for example E6020 (Ishizaka S T et al. “E6020: a syntheticToll-like receptor 4 agonist as a vaccine adjuvant.” Expert Rev.Vaccines. 2007 October; 6 (5):773-84). Various TLR agonists (includingTLR-4 agonists) have been produced and/or sold by, for example, theInfectious Disease Research Institute (IRDI), Corixa, Esai, Avanti PolarLipids, Inc., and Sigma Aldrich. Another exemplary adjuvant thatactivates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate(TDM), and dioctadecyldimethylammonium bromide (DDA). AnotherTLR-activating adjuvant is R848 (resiquimod).

In some embodiments, the adjuvant is or comprises a saponin. Typically,the saponin is a triterpene glycoside, such as those isolated from thebark of the Quillaja saponaria tree. A saponin extract from a biologicalsource can be further fractionated (e.g., by chromatography) to isolatethe portions of the extract with the best adjuvant activity and withacceptable toxicity. Typical fractions of extract from Quillajasaponaria tree used as adjuvants are known as fractions A and C.

A particular form of saponins that may be used in vaccine formulationsdescribed herein is immunostimulating complexes (ISCOMs). ISCOMs are anart-recognized class of adjuvants, that generally comprise Quillajasaponin fractions and lipids (e.g., cholesterol and phospholipids suchas phosphatidyl choline). In certain embodiments, an ISCOM is assembledtogether with a polypeptide or nucleic acid of interest. However,different saponin fractions may be used in different ratios. Inaddition, the different saponin fractions may either exist together inthe same particles or have substantially only one fraction per particle(such that the indicated ratio of fractions A and C are generated bymixing together particles with the different fractions). In thiscontext, “substantially” refers to less than 20%, 15%, 10%, 5%, 4%, 3%,2% or even 1%. Such adjuvants may comprise fraction A and fraction Cmixed into a ratio of 70-95 A:30-5 C, such as 70 A:30 C to 75 A:5 C, 75A:5 C to 80 A:20 C, 80 A:20 C to 85 A:15 C, 85 A:15 C to 90 A:10 C, 90A:10 C to 95 A:5 C, or 95 A:5 C to 99 A:1 C.

In certain embodiments, combinations of adjuvants are used. Threeexemplary combinations of adjuvants are MPL and alum, E6020 and alum,and MPL and an ISCOM.

Adjuvants may be covalently bound to antigens. In some embodiments, theadjuvant may comprise a protein which induces inflammatory responsesthrough activation of antigen-presenting cells (APCs). In someembodiments, one or more of these proteins can be recombinantly fusedwith an antigen of choice, such that the resultant fusion moleculepromotes dendritic cell maturation, activates dendritic cells to producecytokines and chemokines, and ultimately, enhances presentation of theantigen to T cells and initiation of T cell responses (see Wu et al.,Cancer Res 2005; 65 (11), pp 4947-4954). In certain embodiments, apolypeptide described herein is presented in the context of thetrivalent conjugate system, comprising a fusion protein of S. pneumoniaePneumococcal surface adhesin A (PsaA) with the pneumolysoid PdT and acell wall polysaccharide (PsaA:PdT-CPs), described in Lu et al.(“Protection against Pneumococcal colonization and fatal pneumonia by atrivalent conjugate of a fusion protein with the cell wallpolysaccharide.” Infect Immun. 2009 May; 77 (5):2076-83). PdT carriesthree amino acid substitutions (W433F, D385N, and C428G) which renderthe molecule nontoxic but do not interfere with its TLR-4-mediatedinflammatory properties. Conjugation of a polysaccharide to the fusionof a polypeptide to the TLR-4-agonist PdT results in greatly enhancesimmunological response to the polypeptide. In some embodiments, one ormore polypeptides described herein are used in place of PsaA in thetrivalent conjugate. The trivalent conjugate system typically includesalum and is usually administered parenterally. Other exemplary adjuvantsthat may be covalently bound to antigens comprise polysaccharides,pneumolysin, synthetic peptides, lipopeptides, and nucleic acids.

Typically, the same adjuvant or mixture of adjuvants is present in eachdose of a vaccine. Optionally, however, an adjuvant may be administeredwith the first dose of vaccine and not with subsequent doses (i.e.,booster shots). Alternatively, a strong adjuvant may be administeredwith the first dose of vaccine and a weaker adjuvant or lower dose ofthe strong adjuvant may be administered with subsequent doses. Theadjuvant can be administered before the administration of the antigen,concurrent with the administration of the antigen or after theadministration of the antigen to a subject (sometimes within 1, 2, 6, or12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants areappropriate for human patients, non-human animals, or both.

2. Additional Components of a Vaccine or Immunogenic Composition

In addition to the antigens and the adjuvants described above, a vaccineformulation or immunogenic composition may include one or moreadditional components.

In certain embodiments, the vaccine formulation or immunogeniccomposition may include one or more stabilizers such as sugars (such assucrose, glucose, or fructose), phosphate (such as sodium phosphatedibasic, potassium phosphate monobasic, dibasic potassium phosphate, ormonosodium phosphate), glutamate (such as monosodium L-glutamate),gelatin (such as processed gelatin, hydrolyzed gelatin, or porcinegelatin), amino acids (such as arginine, asparagine, histidine,L-histidine, alanine, valine, leucine, isoleucine, serine, threonine,lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine,or sodium borate.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more buffers such as a mixture of sodiumbicarbonate and ascorbic acid. In some embodiments, the vaccineformulation may be administered in saline, such as phosphate bufferedsaline (PBS), or distilled water.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more surfactants such as polysorbate 80(Tween 80), Triton X-100, Polyethylene glycol tert-octylphenyl ethert-Octylphenoxypolyethoxyethanol4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100);Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitanmonolaurate (TWEEN 20); and 4-(1,1,3,3-Tetramethylbutyl)phenol polymerwith formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic ornonionic.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more salts such as sodium chloride, ammoniumchloride, calcium chloride, or potassium chloride.

In certain embodiments, a preservative is included in the vaccine orimmunogenic composition. In other embodiments, no preservative is used.A preservative is most often used in multi-dose vaccine vials, and isless often needed in single-dose vaccine vials. In certain embodiments,the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzylalcohol, and/or sorbic acid.

In certain embodiments, the vaccine formulation or immunogeniccomposition is a controlled release formulation.

E. DNA Vaccines

In certain aspects, the vaccine comprises one or more of the nucleicacids disclosed herein or corresponding to the polypeptides describedherein. When a nucleic acid vaccine is administered to a patient, thecorresponding gene product (such as a desired antigen) is produced inthe patient's body. In some embodiments, nucleic acid vaccine vectorsthat include optimized recombinant polynucleotides can be delivered to amammal (including humans) to induce a therapeutic or prophylactic immuneresponse. The nucleic acid may be, for example, DNA, RNA, or a syntheticnucleic acid. The nucleic acid may be single stranded or doublestranded.

Nucleic acid vaccine vectors (e.g., adenoviruses, liposomes,papillomaviruses, retroviruses, etc.) can be administered directly tothe mammal for transduction of cells in vivo. The nucleic acid vaccinescan be formulated as pharmaceutical compositions for administration inany suitable manner, including parenteral administration. Plasmidvectors are typically more efficient for gene transfer to muscle tissue.The potential to deliver DNA vectors to mucosal surfaces by oraladministration has also been reported (PLGA encapsulated Rotavirus andHepatitis B) and DNA plasmids have been utilized for direct introductionof genes into other tissues. DNA vaccines have been introduced intoanimals primarily by intramuscular injection, by gene gun delivery, orby electroporation. After being introduced, the plasmids are generallymaintained episomally without replication. Expression of the encodedproteins has been shown to persist for extended time periods, providingstimulation of B and T cells.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of an infection or other condition, thephysician evaluates vector toxicities, progression of the disease, andthe production of anti-vector antibodies, if any. Often, the doseequivalent of a naked nucleic acid from a vector is from about 1 μg to 1mg for a typical 70 kilogram patient, and doses of vectors used todeliver the nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses. The toxicity and therapeutic efficacy of the nucleicacid vaccine vectors can be determined using standard pharmaceuticalprocedures in cell cultures or experimental animals.

A nucleic acid vaccine can contain DNA, RNA, a modified nucleic acid, ora combination thereof. In some embodiments, the vaccine comprises one ormore cloning or expression vectors; for instance, the vaccine maycomprise a plurality of expression vectors each capable of autonomousexpression of a nucleotide coding region in a mammalian cell to produceat least one immunogenic polypeptide. An expression vector oftenincludes a eukaryotic promoter sequence, such as the nucleotide sequenceof a strong eukaryotic promoter, operably linked to one or more codingregions. The compositions and methods herein may involve the use of anyparticular eukaryotic promoter, and a wide variety are known; such as aCMV or RSV promoter. The promoter can be heterologous with respect tothe host cell. The promoter used may be a constitutive promoter.

A vector useful in the present compositions and methods can be circularor linear, single-stranded or double stranded and can be a plasmid,cosmid, or episome. In a suitable embodiment, each nucleotide codingregion is on a separate vector; however, it is to be understood that oneor more coding regions can be present on a single vector, and thesecoding regions can be under the control of a single or multiplepromoters.

Numerous plasmids may be used for the production of nucleic acidvaccines. Suitable embodiments of the nucleic acid vaccine employconstructs using the plasmids VR1012 (Vical Inc., San Diego Calif.),pCMVI.UBF3/2 (S. Johnston, University of Texas) or pcDNA3.1 (InVitrogenCorporation, Carlsbad, Calif.) as the vector. In addition, the vectorconstruct can contain immunostimulatory sequences (ISS), such asunmethylated dCpG motifs, that stimulate the animal's immune system. Thenucleic acid vaccine can also encode a fusion product containing theimmunogenic polypeptide. Plasmid DNA can also be delivered usingattenuated bacteria as delivery system, a method that is suitable forDNA vaccines that are administered orally. Bacteria are transformed withan independently replicating plasmid, which becomes released into thehost cell cytoplasm following the death of the attenuated bacterium inthe host cell.

DNA vaccines, including the DNA encoding the desired antigen, can beintroduced into a host cell in any suitable form including, the fragmentalone, a linearized plasmid, a circular plasmid, a plasmid capable ofreplication, an episome, RNA, etc. Preferably, the gene is contained ina plasmid. In certain embodiments, the plasmid is an expression vector.Individual expression vectors capable of expressing the genetic materialcan be produced using standard recombinant techniques. See e.g.,Maniatis et al., 1985 Molecular Cloning: A Laboratory Manual or DNACloning, Vol. I and II (D. N. Glover, ed., 1985) for general cloningmethods.

Routes of administration include, but are not limited to, intramuscular,intranasal, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Typical routes of administration include intramuscular, intraperitoneal,intradermal and subcutaneous injection. Genetic constructs may beadministered by means including, but not limited to, traditionalsyringes, needleless injection devices, “microprojectile bombardmentgene guns”, or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound. DNA vaccines can be delivered byany method that can be used to deliver DNA as long as the DNA isexpressed and the desired antigen is made in the cell.

In some embodiments, a DNA vaccine is delivered via known transfectionreagents such as cationic liposomes, fluorocarbon emulsion, cochleate,tubules, gold particles, biodegradable microspheres, or cationicpolymers. Cochleate delivery vehicles are stable phospholipid calciumprecipitants consisting of phosphatidyl serine, cholesterol, andcalcium; this nontoxic and noninflammatory transfection reagent can bepresent in a digestive system. Biodegradable microspheres comprisepolymers such as poly(lactide-co-glycolide), a polyester that can beused in producing microcapsules of DNA for transfection. Lipid-basedmicrotubes often consist of a lipid of spirally wound two layers packedwith their edges joined to each other. When a tubule is used, thenucleic acid can be arranged in the central hollow part thereof fordelivery and controlled release into the body of an animal.

In some embodiments, DNA vaccine is delivered to mucosal surfaces viamicrospheres. Bioadhesive microspheres can be prepared using differenttechniques and can be tailored to adhere to any mucosal tissue includingthose found in eye, nasal cavity, urinary tract, colon andgastrointestinal tract, offering the possibilities of localized as wellas systemic controlled release of vaccines. Application of bioadhesivemicrospheres to specific mucosal tissues can also be used for localizedvaccine action. In some embodiments, an alternative approach for mucosalvaccine delivery is the direct administration to mucosal surfaces of aplasmid DNA expression vector which encodes the gene for a specificprotein antigen.

The DNA plasmid vaccines according to the present invention areformulated according to the mode of administration to be used. In someembodiments where DNA plasmid vaccines are injectable compositions, theyare sterile, and/or pyrogen free and/or particulate free. In someembodiments, an isotonic formulation is preferably used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some embodiments, isotonic solutionssuch as phosphate buffered saline are preferred. In some embodiments,stabilizers include gelatin and albumin. In some embodiments, avasoconstriction agent is added to the formulation. In some embodiments,a stabilizing agent that allows the formulation to be stable at room orambient temperature for extended periods of time, such as LGS or otherpolycations or polyanions is added to the formulation.

In some embodiments, the DNA vaccine may further comprises apharmacologically acceptable carrier or diluent. Suitable carriers forthe vaccine are well known to those skilled in the art and include butare not limited to proteins, sugars, etc. Such carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous carriers are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and antimicrobials include antioxidants,chelating agents, inert gases and the like. Preferred preservativesinclude formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

An alternative approach to delivering the nucleic acid to an animalinvolves the use of a viral or bacterial vector. Examples of suitableviral vectors include adenovirus, polio virus, pox viruses such asvaccinia, canary pox, and fowl pox, herpes viruses, including catfishherpes virus, adenovirus-associated vector, and retroviruses. Exemplarybacterial vectors include attenuated forms of Salmonella, Shigella,Edwardsiella ictaluri, Yersinia ruckerii, and Listeria monocytogenes. Insome embodiments, the nucleic acid is a vector, such as a plasmid, thatis capable of autologous expression of the nucleotide sequence encodingthe immunogenic polypeptide.

F. Use of Vaccines

The S. pneumoniae vaccines described herein may be used for prophylacticand/or therapeutic treatment of S. pneumoniae. Accordingly, thisapplication provides a method for treating a subject suffering from orsusceptible to S. pneumoniae infection, comprising administering aneffective amount of any of the vaccine formulations described herein. Insome aspects, the method inhibits S. pneumoniae colonization in anindividual. In some aspects, the method inhibits S. pneumoniae symptomsor sequelae, such as sepsis. The subject receiving the vaccination maybe a male or a female, and may be a child or adult. In some embodiments,the subject being treated is a human. In other embodiments, the subjectis a non-human animal.

1. Prophylactic Use

In prophylactic embodiments, the vaccine is administered to a subject toinduce an immune response that can help protect against theestablishment of S. pneumoniae, for example by protecting againstcolonization, the first and necessary step in disease. Thus, in someaspects, the method inhibits infection by S. pneumoniae in anon-colonized or uninfected subject. In another aspect, the method mayreduce the duration of colonization in an individual who is alreadycolonized.

In some embodiments, the vaccine compositions of the invention conferprotective immunity, allowing a vaccinated individual to exhibit delayedonset of symptoms or sequelae, or reduced severity of symptoms orsequelae, as the result of his or her exposure to the vaccine. Incertain embodiments, the reduction in severity of symptoms or sequelaeis at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%. In particularembodiments, vaccinated individuals may display no symptoms or sequelaeupon contact with S. pneumoniae, do not become colonized by S.pneumoniae, or both. Protective immunity is typically achieved by one ormore of the following mechanisms: mucosal, humoral, or cellularimmunity. Mucosal immunity is primarily the result of secretory IgA(sIGA) antibodies on mucosal surfaces of the respiratory,gastrointestinal, and genitourinary tracts. The sIGA antibodies aregenerated after a series of events mediated by antigen-processing cells,B and T lymphocytes, that result in sIGA production by B lymphocytes onmucosa-lined tissues of the body. Humoral immunity is typically theresult of IgG antibodies and IgM antibodies in serum. Cellular immunitycan be achieved through cytotoxic T lymphocytes or through delayed-typehypersensitivity that involves macrophages and T lymphocytes, as well asother mechanisms involving T cells without a requirement for antibodies.In particular, cellular immunity may be mediated by T_(H)1 or T_(H)17cells.

Essentially any individual has a certain risk of becoming infected withS. pneumoniae. However, certain sub-populations have an increased riskof infection. In some embodiments, a vaccine formulation as describedherein (e.g., a composition comprising one or more polypeptides fromTable 1 or 2, or nucleic acids encoding the polypeptides, or antibodiesreactive with the polypeptides) is administered to patients that areimmunocompromised.

An immunocompromising condition arising from a medical treatment islikely to expose the individual in question to a higher risk ofinfection with S. pneumoniae. It is possible to treat an infectionprophylactically in an individual having the immunocompromised conditionbefore or during treatments known to compromise immune function. Byprophylactically treating with an antigenic composition (e.g., two ormore antigens from Table 1 or 2, or nucleic acids encoding theantigens), or with antibodies reactive to two or more antigens fromTable 1 or 2, before or during a treatment known to compromise immunefunction, it is possible to prevent a subsequent S. pneumoniae infectionor to reduce the risk of the individual contracting an infection due tothe immunocompromised condition. Should the individual contract an S.pneumoniae infection e.g., following a treatment leading to animmunocompromised condition it is also possible to treat the infectionby administering to the individual an antigen composition.

The following groups are at increased risk of pneumococcal disease orits complications, and therefore it is advantageous for subjects fallinginto one or more of these groups to receive a vaccine formulationdescribed herein: children, especially those from 1 month to 5 years oldor 2 months to 2 years old; children who are at least 2 years of agewith asplenia, splenic dysfunction or sickle-cell disease; children whoare at least 2 years of age with nephrotic syndrome, chroniccerebrospinal fluid leak, HIV infection or other conditions associatedwith immunosuppression.

In another embodiment, at least one dose of the pneumococcal antigencomposition is given to adults in the following groups at increased riskof pneumococcal disease or its complications: all persons 65 years ofage; adults with asplenia, splenic dysfunction or sickle-cell disease;adults with the following conditions: chronic cardiorespiratory disease,cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome,diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDSand other conditions associated with immunosuppression (Hodgkin'sdisease, lymphoma, multiple myeloma, immunosuppression for organtransplantation), individuals with cochlear implants; individuals withlong-term health problems such as heart disease and lung disease, aswell as individuals who are taking any drug or treatment that lowers thebody's resistance to infection, such as long-term steroids, certaincancer drugs, radiation therapy; Alaskan natives and certain NativeAmerican populations.

2. Therapeutic Use

In therapeutic applications, the vaccine may be administered to apatient suffering from S. pneumoniae infection, in an amount sufficientto treat the patient. Treating the patient, in this case, refers toreducing S. pneumoniae symptoms and/or bacterial load and/or sequelaebin an infected individual. In some embodiments, treating the patientrefers to reducing the duration of symptoms or sequelae, or reducing theintensity of symptoms or sequelae. In some embodiments, the vaccinereduces transmissibility of S. pneumoniae from the vaccinated patient.In certain embodiments, the reductions described above are at least 25%,30%, 40%, 50%, 60%, 70%, 80% or even 90%.

In therapeutic embodiments, the vaccine is administered to an individualpost-infection. The vaccine may be administered shortly after infection,e.g. before symptoms or sequelae manifest, or may be administered duringor after manifestation of symptoms or sequelae.

A therapeutic S. pneumoniae vaccine can reduce the intensity and/orduration of the various symptoms or sequelae of S. pneumoniae infection.Symptoms or sequelae of S. pneumoniae infection can take many forms. Insome cases, an infected patient develops pneumonia, acute sinusitis,otitis media (ear infection), meningitis, bacteremia, sepsis,osteomyelitis, septic arthritis, endocarditis, peritonitis,pericarditis, cellulitis, or brain abscess.

Sepsis is a rare but life-threatening complication of S. pneumoniaeinfection, where the bacterium invades the bloodstream and systemicinflammation results. Typically, fever is observed and white blood cellcount increases. A further description of sepsis is found in Goldstein,B. et al. “International pediatric sepsis consensus conference:definitions for sepsis and organ dysfunction in pediatrics.” PediatrCrit. Care Med. January 2005; 6 (1):2-8.

3. Assaying Vaccination Efficacy

The efficacy of vaccination with the vaccines disclosed herein may bedetermined in a number of ways, in addition to the clinical outcomesdescribed above. First, one may assay IL-17 levels (particularly IL-17A)by stimulating T cells derived from the subject after vaccination. TheIL-17 levels may be compared to IL-17 levels in the same subject beforevaccination. Increased IL-17 (e.g., IL-17A) levels, such as a 1.5 fold,2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase,would indicate an increased response to the vaccine. Alternatively (orin combination), one may assay neutrophils in the presence of T cells orantibodies from the patient for pneumococcal killing. Increasedpneumococcal killing, such as a 1.5 fold, 2-fold, 5-fold, 10-fold,20-fold, 50-fold or 100-fold or more increase, would indicate anincreased response to the vaccine. In addition, one may measure T_(H)17cell activation, where increased T_(H)17 cell activation, such as a 1.5fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or moreincrease, correlates with an increased response to the vaccine. One mayalso measure levels of an antibody specific to the vaccine, whereincreased levels of the specific antibody, such as a 1.5 fold, 2-fold,5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, arecorrelated with increased vaccine efficacy. In certain embodiments, twoor more of these assays are used. For example, one may measure IL-17levels and the levels of vaccine-specific antibody. Alternatively, onemay follow epidemiological markers such as incidence of, severity of, orduration of pneumococcal infection in vaccinated individuals compared tounvaccinated individuals.

Vaccine efficacy may also be assayed in various model systems such asthe mouse model. For instance, BALB/c or C57BL/6 strains of mice may beused. After administering the test vaccine to a subject (as a singledose or multiple doses), the experimenter administers a challenge doseof S. pneumoniae. In some cases, a challenge dose administeredintranasally is sufficient to cause S. pneumoniae colonization(especially nasal colonization) in an unvaccinated animal, and in somecases a challenge dose administered via aspiration is sufficient tocause sepsis and a high rate of lethality in unvaccinated animals. Onecan then measure the reduction in colonization or the reduction inlethality in vaccinated animals. Examples 5-8 and 10 show the efficacyof polypeptides of Table 1 in inhibiting S. pneumoniae nasalcolonization following intranasal challenge in the mouse model. Examples11 and 12 show the efficacy of polypeptides of Table 1 in protectingagainst sepsis and death following infection with S. pneumoniae viaaspiration in the mouse model.

G. Use of Immunogenic Compositions

1. Defense Against S. pneumoniae Infection

The immunogenic compositions of the present disclosure are designed toelicit an immune response against S. pneumoniae. Compositions describedherein (e.g., ones comprising one or more polypeptides of Table 1 or 2,or nucleic acids encoding the polypeptides) may stimulate an antibodyresponse or a cell-mediated immune response, or both, in the mammal towhich it is administered. In some embodiments, the compositionstimulates a T_(H)1-biased CD4⁺ T cell response, a T_(H)17-biased CD4⁺ Tcell response and/or a CD8⁺ T cell response. In some embodiments, thecomposition stimulates an antibody response. In some embodiments, thecomposition stimulates a T_(H)1-biased CD4⁺ T cell response,T_(H)17-biased CD4⁺ T cell response and/or a CD8⁺ T cell response, andan antibody response.

In certain embodiments, the composition (e.g., one comprising one ormore polypeptides of Table 1 or 2, or nucleic acids encoding thepolypeptides, or antibodies reactive with the peptides) includes acytokine or nucleotide coding region encoding a cytokine such as IL-17,to provide additional stimulation to the immune system of the mammal. Incertain embodiments, the composition comprises a cytokine such as IL-17.

While not wishing to be bound by theory, in some embodiments a T_(H)17cell response is desirable in mounting an immune response to thecompositions disclosed herein, e.g., ones comprising one or morepolypeptides of Table 1 or 2. In certain embodiments, an active T_(H)17response is beneficial in clearing a pneumococcal infection. Forinstance, mice lacking the IL-17A receptor show decreased whole cellvaccine-based protection from a pneumococcal challenge (Lu et al.,“Interleukin-17A mediates acquired immunity to pneumococcalcolonization.” PLoS Pathog. 2008 Sep. 19; 4 (9)).

Thus, herein is provided a method of increasing IL-17 production byadministering the compositions described herein (e.g., ones comprisingone or more polypeptides of Table 1 or 2) to a subject. Furthermore,this application provides a method of activating T_(H)17 cells byadministering said compositions to a subject. In certain embodiments,increased IL-17A levels result in increased pneumococcal killing byneutrophils or neutrophil-like cells, for instance by inducingrecruitment and activation of neutrophils of neutrophil-like cells. Incertain embodiments, this pneumococcal killing is independent ofantibodies and complement. However, specific antibody production andcomplement activation may be useful additional mechanisms thatcontribute to clearing of a pneumococcal infection.

Immunogenic compositions containing immunogenic polypeptides orpolynucleotides encoding immunogenic polypeptides together with apharmaceutical carrier are also provided.

In some instances, the immunogenic composition comprises one or morenucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13,265, 266 and 267, such as one or more nucleic acids selected from SEQ IDNos. 24-31, 271, 272 and 273. In some embodiments these nucleic acidsare expressed in the immunized individual, producing the encoded S.pneumoniae antigens, and the S. pneumoniae antigens so produced canproduce an immunostimulatory effect in the immunized individual.

Such a nucleic acid-containing immunostimulatory composition maycomprise, for example, an origin of replication, and a promoter thatdrives expression of one or more nucleic acids encoding one or morepolypeptides of SEQ ID NOS: 1-13, 265, 266 and 267. Such a compositionmay also comprise a bacterial plasmid vector into which is inserted apromoter (sometimes a strong viral promoter), one or more nucleic acidsencoding one or more polypeptides of SEQ ID NOS: 1-13, 265, 266 and 267,and a polyadenylation/transcriptional termination sequence. In someinstances, the nucleic acid is DNA.

H. Diagnostic Uses

This application provides, inter alia, a rapid, inexpensive, sensitive,and specific method for detection of S. pneumoniae in patients. In thisrespect it should be useful to all hospitals and physicians examiningand treating patients with or at risk for S. pneumoniae infection.Detection kits can be simple enough to be set up in any local hospitallaboratory, and the antibodies and antigen-binding portions thereof canreadily be made available to all hospitals treating patients with or atrisk for S. pneumoniae infection. As used herein, “patient” refers to anindividual (such as a human) that either has an S. pneumoniae infectionor has the potential to contract an S. pneumoniae infection. A patientmay be an individual (such as a human) that has an S. pneumoniaeinfection, has the potential to contract an S. pneumoniae infection, whohas recovered from S. pneumoniae infection, and/or an individual whoseinfection status is unknown.

In some embodiments, one may perform a diagnostic assay using two ormore antibodies, each of which binds one of the antigens of Table 1 or 2to detect S. pneumoniae in an individual. In some embodiment, one of theantigens is SEQ ID NO: 265, 266, or 268. The instant disclosure alsoprovides a method of phenotyping biological samples from patientssuspected of having a S. pneumoniae infection: (a) obtaining abiological sample from a patient; (b) contacting the sample with two ormore S. pneumoniae-specific antibodies or antigen-binding portionsthereof under conditions that allow for binding of the antibody orantigen-binding portion to an epitope of S. pneumoniae; where bindingindicates the presence of S. pneumoniae in the sample. In someembodiments, the binding to the biological sample is compared to bindingof the same antibody to a negative control tissue, wherein if thebiological sample shows the presence of S. pneumoniae as compared to thenegative control tissue, the patient is identified as likely having a S.pneumoniae infection. In some cases, binding of one antibody indicatesthe presence of S. pneumoniae; in other cases, the binding of two ormore antibodies indicates the presence of S. pneumoniae. Theaforementioned test may be appropriately adjusted to detect otherbacterial infections, for instance by using an antibody immunoreactive ahomolog (from another bacterial species) of one of the proteinsdescribed in Table 1. In some embodiments, the antibodies raised againsta S. pneumoniae protein in Table 1 or 2 will also bind the homolog inanother Streptococcus species, especially if the homologs have a highpercentage sequence identity.

Alternatively, one may use an antigen of Table 1 or 2 (such as SEQ IDNO: 265, 266, or 268) to detect anti-S. pneumoniae antibodies in anindividual. The instant disclosure also provides a method of phenotypingbiological samples from patients suspected of having a S. pneumoniaeinfection: (a) obtaining a biological sample from a patient; (b)contacting the sample with two or more S. pneumoniae-specific antigensselected from Table 1 or 2 or portions thereof under conditions thatallow for binding of the antigen (or portion thereof) to any hostantibodies present in the sample; where binding indicates the presenceof anti-S. pneumoniae antibodies in the sample. In some embodiments, thebinding to the biological sample is compared to binding of the sameantigen to a negative control tissue, wherein if the biological sampleshows the presence of anti-S. pneumoniae antibodies as compared to thenegative control tissue, the patient is identified as likely either (1)having a S. pneumoniae infection, or (2) having had a S. pneumoniaeinfection in the past. In some cases, detecting one antibody indicates acurrent or past infection with S. pneumoniae; in other cases, detectingtwo or more antibodies indicates a current or past infection with S.pneumoniae. The aforementioned test may be appropriately adjusted todetect other bacterial infections, for instance by using a homolog (fromanother bacterial species (e.g., a Streptococcal species) of theproteins described in Table 1.

In some embodiments, the immune cell response of a mammalian cell may bequantified ex vivo. A method for such quantification comprisesadministering the compositions herein disclosed to a mammalian T cell exvivo, and quantifying the change in cytokine production of the mammalianT cell in response to the composition. In these methods, the cytokinemay be, for example, IL-17.

The binding of an S. pneumoniae antibody to an antigen (e.g., apolypeptide of Table 1 or 2, such as SEQ ID NO: 265, 266, or 268) may bemeasured using any appropriate method. Such methods include ELISA(enzyme-linked immunosorbent assay), Western blotting, competitionassay, and spot-blot. The detection step may be, for instance,chemiluminescent, fluorescent, or colorimetric. One suitable method formeasuring antibody-protein binding is the Luminex xMAP system, wherepeptides are bound to a dye-containing microsphere. Certain systems,including the xMAP system, are amenable to measuring several differentmarkers in multiplex, and could be used to measure levels of antibodiesat once. In some embodiments, other systems are used to assay aplurality of markers in multiplex. For example, profiling may beperformed using any of the following systems: antigen microarrays, beadmicroarrays, nanobarcodes particle technology, arrayed proteins fromcDNA expression libraries, protein in situ array, protein arrays ofliving transformants, universal protein array, lab-on-a-chipmicrofluidics, and peptides on pins. Another type of clinical assay is achemiluminescent assay to detect antibody binding. In some such assays,including the VITROS Eci anti-HCV assay, antibodies are bound to asolid-phase support made up of microparticles in liquid suspension, anda surface fluorometer is used to quantify the enzymatic generation of afluorescent product.

In some embodiments, if the biological sample shows the presence of S.pneumoniae (e.g., by detecting one or more polypeptide of Table 1 or 2,such as SEQ ID NO: 265, 266, or 268, or an antibody that binds one ofsaid polypeptides), one may administer a therapeutically effectiveamount of the compositions and therapies described herein to thepatient. The biological sample may comprise, for example, blood, semen,urine, vaginal fluid, mucus, saliva, feces, urine, cerebrospinal fluid,or a tissue sample. In some embodiments, the biological sample is anorgan intended for transplantation. In certain embodiments, before thedetection step, the biological sample is subject to culture conditionsthat promote the growth of S. pneumoniae.

The diagnostic tests herein (e.g., those that detect a polypeptide ofTable 1 or 2, such as SEQ ID NO: 265, 266, or 268, or an antibody thatbinds one of said polypeptides) may be used to detect S. pneumoniae in avariety of samples, including samples taken from patients and samplesobtained from other sources. For example, the diagnostic tests may beused to detect S. pneumoniae in food, drink, or ingredients for food anddrink; on objects such as medical instruments, medical devices such ascochlear implants and pacemakers, shoes, clothing, furniture includinghospital furniture, and drapes including hospital drapes; or in samplestaken from the environment such as plant samples. In some embodiments,the tests herein may be performed on samples taken from animals such asagricultural animals (cows, pigs, chickens, goats, horses and the like),companion animals (dogs, cats, birds, and the like), or wild animals. Incertain embodiments, the tests herein may be performed on samples takenfrom cell cultures such as cultures of human cells that produce atherapeutic protein, cultures of bacteria intended to produce a usefulbiological molecule, or cultures of cells grown for research purposes.

This disclosure also provides a method of determining the location of aS. pneumoniae infection in a patient comprising: (a) administering apharmaceutical composition comprising a labeled S. pneumoniae antibodyor antigen-binding portion thereof to the patient, and (b) detecting thelabel, wherein binding indicates a S. pneumoniae infection in aparticular location in the patient. Such a diagnostic may also comprisecomparing the levels of binding in the patient to a control. In certainembodiments, the method further comprises, if the patient has a S.pneumoniae infection, treating the infection by administering atherapeutically effective amount of a S. pneumoniae-binding antibody orantigen-binding portion thereof to the patient. In certain embodiments,the method further comprises, if the patient has a S. pneumoniaeinfection, treating the infection by administering a therapeuticallyeffective amount of a S. pneumoniae protein of Table 1 or 2, orimmunogenic portion thereof, to the patient. The method may furthercomprise determining the location and/or volume of the S. pneumoniae inthe patient. This method may be used to evaluate the spread of S.pneumoniae in the patient and determine whether a localized therapy isappropriate.

In some embodiments, the anti-S. pneumoniae antibodies or T cellsdescribed herein may be used to make a prognosis of the course ofinfection. In some embodiments, the anti-S. pneumoniae antibodies or Tcells herein may be detected in a sample taken from a patient. Ifantibodies or T cells are present at normal levels, it would indicatethat the patient has raised an immune response against anti-S.pneumoniae. If antibodies or T cells are absent, or present at reducedlevels, it would indicate that the patient is failing to raise asufficient response against anti-S. pneumoniae, and a more aggressivetreatment would be recommended. In some embodiments, antibodies or Tcells present at reduced levels refers to antibodies that are present atless than 50%, 20%, 10%, 5%, 2%, or 1% the level of antibodies or Tcells typical in a patient with a normal immune system. Antibodies maybe detected by affinity for any of the antigens described herein (e.g.,those in Table 1 and/or 2), for example using ELISA. T cells may bedetected by ex vivo responses for any of the antigens described herein(e.g., those in Table 1 and/or 2), for example using ELISA or ELISPOTassays.

In some embodiments, detection of specific S. pneumoniae antigens (e.g.,those in Table 1 and/or 2, such as SEQ ID NO: 265, 266, or 268) may beused to predict the progress and symptoms of S. pneumoniae infection ina patient. It will be understood by one of skill in the art that themethods herein are not limited to detection of S. pneumoniae. Otherembodiments include the detection of related bacteria including bacteriawith proteins homologous to the proteins described in Table 1 or 2. Suchrelated bacteria include, for example, other strains of Streptococcus.

I. Doses and Routes of Administration

1. Dosage Forms, Amounts, and Timing

The amount of antigen in each vaccine or immunogenic composition dose isselected as an effective amount, which induces a prophylactic ortherapeutic response, as described above, in either a single dose orover multiple doses. Preferably, the dose is without significant adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific antigen is employed. Generally, it is expected that adose will comprise 1-1000 μg of each protein, in some instances 2-100μg, for instance 4-40 μg. In some aspects, the vaccine formulationcomprises 1-1000 μg of the polypeptide and 1-250 μg of the adjuvant. Insome embodiments, the appropriate amount of antigen to be delivered willdepend on the age, weight, and health (e.g. immunocompromised status) ofa subject. When present, typically an adjuvant will be present inamounts from 1 μg-250 μg per dose, for example 50-150 μg, 75-125 μg or100 μg.

In some embodiments, only one dose of the vaccine is administered toachieve the results described above. In other embodiments, following aninitial vaccination, subjects receive one or more boost vaccinations,for a total of two, three, four or five vaccinations. Advantageously,the number is three or fewer. A boost vaccination may be administered,for example, about 1 month, 2 months, 4 months, 6 months, or 12 monthsafter the initial vaccination, such that one vaccination regimeninvolves administration at 0, 0.5-2 and 4-8 months. It may beadvantageous to administer split doses of vaccines which may beadministered by the same or different routes.

The vaccines and immunogenic compositions described herein may take on avariety of dosage forms. In certain embodiments, the composition isprovided in solid or powdered (e.g., lyophilized) form; it also may beprovided in solution form. In certain embodiments, a dosage form isprovided as a dose of lyophilized composition and at least one separatesterile container of diluent.

In some embodiments, the composition will be administered in a doseescalation manner, such that successive administrations of thecomposition contain a higher concentration of composition than previousadministrations. In some embodiments, the composition will beadministered in a manner such that successive administrations of thecomposition contain a lower concentration of composition than previousadministrations.

In therapeutic applications, compositions are administered to a patientsuffering from a disease in an amount sufficient to treat the patient.Therapeutic applications of a composition described herein includereducing transmissibility, slowing disease progression, reducingbacterial viability or replication, or inhibiting the expression ofproteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%,30%, 20% or 10% of the levels at which they would occur in individualswho are not treated with the composition.

In prophylactic embodiments, compositions are administered to a human orother mammal to induce an immune response that can inhibit theestablishment of an infectious disease or other condition. In someembodiments, a composition may partially block the bacterium fromestablishing an infection.

In some embodiments, the compositions are administered in combinationwith antibiotics. This co-administration is particularly appropriatewhen the pharmaceutical composition is administered to a patient who hasrecently been exposed (or is suspected of having been recently exposed)to S. pneumoniae. Many antibiotics are used to treat pneumococcalinfections, including penicillin, amoxicillin, amoxicillin/clavulanate,cefuroxime, cefotaxime, ceftriaxone, and vancomycin. The appropriateantibiotic may be selected based on the type and severity of theinfection, as well as any known antibiotic resistance of the infection(Jacobs M R “Drug-resistant Streptococcus pneumoniae: rationalantibiotic choices” Am J. Med. 1999 May 3; 106 (5A):19S-25S).

2. Routes of Administration

The vaccine formulations and pharmaceutical compositions herein can bedelivered by administration to an individual, typically by systemicadministration (e.g., intravenous, intraperitoneal, intramuscular,intradermal, subcutaneous, subdermal, transdermal, intracranial,intranasal, mucosal, anal, vaginal, oral, buccal route or they can beinhaled) or they can be administered by topical application. In someembodiments, the route of administration is intramuscular. In otherembodiments, the route of administration is subcutaneous. In yet otherembodiments, the route of administration is mucosal. In certainembodiments, the route of administration is transdermal or intradermal

Certain routes of administration are particularly appropriate forvaccine formulations and immunogenic compositions comprising specifiedadjuvants. In particular, transdermal administration is one suitableroute of administration for S. pneumoniae vaccines comprising toxins(e.g. cholera toxin or labile toxin); in other embodiments, theadministration is intranasal. Vaccines formulated with Alphavirusreplicons may be administered, for example, by the intramuscular or thesubcutaneous route. Vaccines comprising Monophosphory Lipid A (MPL),Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide(DDA) are suitable (inter alia) for intramuscular and subcutaneousadministration. A vaccine comprising resiquimod may be administeredtopically or subcutaneously, for example.

3. Formulations

The vaccine formulation or immunogenic composition may be suitable foradministration to a human patient, and vaccine or immunogeniccomposition preparation may conform to USFDA guidelines. In someembodiments, the vaccine formulation or immunogenic composition issuitable for administration to a non-human animal. In some embodiments,the vaccine or immunogenic composition is substantially free of eitherendotoxins or exotoxins. Endotoxins may include pyrogens, such aslipopolysaccharide (LPS) molecules. The vaccine or immunogeniccomposition may also be substantially free of inactive protein fragmentswhich may cause a fever or other side effects. In some embodiments, thecomposition contains less than 1%, less than 0.1%, less than 0.01%, lessthan 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/orinactive protein fragments. In some embodiments, the vaccine orimmunogenic composition has lower levels of pyrogens than industrialwater, tap water, or distilled water. Other vaccine or immunogeniccomposition components may be purified using methods known in the art,such as ion-exchange chromatography, ultrafiltration, or distillation.In other embodiments, the pyrogens may be inactivated or destroyed priorto administration to a patient. Raw materials for vaccines, such aswater, buffers, salts and other chemicals may also be screened anddepyrogenated. All materials in the vaccine may be sterile, and each lotof the vaccine may be tested for sterility. Thus, in certain embodimentsthe endotoxin levels in the vaccine fall below the levels set by theUSFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecalinjectable composition; 5 EU/kg of product for a non-intrathecalinjectable composition, and 0.25-0.5 EU/mL for sterile water.

In certain embodiments, the preparation comprises less than 50%, 20%,10%, or 5% (by dry weight) contaminating protein. In certainembodiments, the desired molecule is present in the substantial absenceof other biological macromolecules, such as other proteins (particularlyother proteins which may substantially mask, diminish, confuse or alterthe characteristics of the component proteins either as purifiedpreparations or in their function in the subject reconstituted mixture).In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dryweight) of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). In someembodiments, the vaccine or immunogenic composition comprising purifiedsubunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% ofprotein from host cells in which the subunit proteins were expressed,relative to the amount of purified subunit. In some embodiments, thedesired polypeptides are substantially free of nucleic acids and/orcarbohydrates. For instance, in some embodiments, the vaccine orimmunogenic composition contains less than 5%, less than 2%, less than1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNAand/or RNA. In certain embodiments, at least 80%, 90%, 95%, 99%, or99.8% (by dry weight) of biological macromolecules of the same type arepresent in the preparation (but water, buffers, and other smallmolecules, especially molecules having a molecular weight of less than5000, can be present).

It is preferred that the vaccine or immunogenic composition has low orno toxicity, within a reasonable risk-benefit ratio. In certainembodiments, the vaccine or immunogenic composition comprisesingredients at concentrations that are less than LD₅₀ measurements forthe animal being vaccinated. LD₅₀ measurements may be obtained in miceor other experimental model systems, and extrapolated to humans andother animals. Methods for estimating the LD₅₀ of compounds in humansand other animals are well-known in the art. A vaccine formulation orimmunogenic composition, and any component within it, might have an LD₅₀value in rats of greater than 100 g/kg, greater than 50 g/kg, greaterthan 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20mg/kg, or greater than 10 mg/kg. A vaccine formulation or immunogeniccomposition that comprises a toxin such as botulinum toxin (which can beused as an adjuvant) should contain significantly less than the LD₅₀ ofbotulinum toxin.

The formulations suitable for introduction of the vaccine formulationsor pharmaceutical composition vary according to route of administration.Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, intranasal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampoules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. In thecase of adoptive transfer of therapeutic T cells, the cells can beadministered intravenously or parenterally.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the polypeptides or packagednucleic acids suspended in diluents, such as water, saline or PEG 400;(b) capsules, sachets or tablets, each containing a predetermined amountof the active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art. The pharmaceutical compositionscan be encapsulated, e.g., in liposomes, or in a formulation thatprovides for slow release of the active ingredient.

The antigens, alone or in combination with other suitable components,can be made into aerosol formulations (e.g., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like. Aerosol formulations can be deliveredorally or nasally.

Suitable formulations for vaginal or rectal administration include, forexample, suppositories, which consist of the polypeptides or packagednucleic acids with a suppository base. Suitable suppository basesinclude natural or synthetic triglycerides or paraffin hydrocarbons. Inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the polypeptides or packaged nucleic acidswith a base, including, for example, liquid triglycerides, polyethyleneglycols, and paraffin hydrocarbons.

J. Preparation and Storage of Vaccine Formulations and ImmunogenicCompositions

The S. pneumoniae vaccines and immunogenic compositions described hereinmay be produced using a variety of techniques. For example, apolypeptide may be produced using recombinant DNA technology in asuitable host cell. A suitable host cell may be bacterial, yeast,mammalian, or other type of cell. The host cell may be modified toexpress an exogenous copy of one of the relevant polypeptide genes.Typically, the gene is operably linked to appropriate regulatorysequences such as a strong promoter and a polyadenylation sequence. Insome embodiments, the promoter is inducible or repressible. Otherregulatory sequences may provide for secretion or excretion of thepolypeptide of interest or retention of the polypeptide of interest inthe cytoplasm or in the membrane, depending on how one wishes to purifythe polypeptide. The gene may be present on an extrachromosomal plasmid,or may be integrated into the host genome. One of skill in the art willrecognize that it is not necessary to use a nucleic acid 100% identicalto the naturally-occurring sequence. Rather, some alterations to thesesequences are tolerated and may be desirable. For instance, the nucleicacid may be altered to take advantage of the degeneracy of the geneticcode such that the encoded polypeptide remains the same. In someembodiments, the gene is codon-optimized to improve expression in aparticular host. The nucleic acid may be produced, for example, by PCRor by chemical synthesis.

Once a recombinant cell line has been produced, a polypeptide may beisolated from it. The isolation may be accomplished, for example, byaffinity purification techniques or by physical separation techniques(e.g., a size column).

In a further aspect of the present disclosure, there is provided amethod of manufacture comprising mixing one or more polypeptides or animmunogenic fragment or variant thereof with a carrier and/or anadjuvant.

In some embodiments, antigens for inclusion the vaccine formulations andimmunogenic compositions may be produced in cell culture. One methodcomprises providing one or more expression vectors and cloningnucleotides encoding one or more polypeptides selected from polypeptideshaving an amino acid sequence of Table 1 or 2, such as SEQ ID NO: 265,266, or 268, then expressing and isolating the polypeptides.

The immunogenic polypeptides described herein, and nucleic acidcompositions that express the polypeptides, can be packaged in packs,dispenser devices, and kits for administering nucleic acid compositionsto a mammal. For example, packs or dispenser devices that contain one ormore unit dosage forms are provided. Typically, instructions foradministration of the compounds will be provided with the packaging,along with a suitable indication on the label that the compound issuitable for treatment of an indicated condition, such as thosedisclosed herein.

V. EXAMPLES Example 1 Antigen Identification and Pooled Murine Screens

Each open reading frame predicted in the S. pneumoniae TIGR4 genome wascloned into an expression vector comprising a tag that is able to bepresented by the major histocompatibility complex (MHC). Each constructwas then expressed in E. coli, and full-length expression validated by asurrogate assay that identifies the tag in the context of MHC. Thescreen is described in more detail in International Application WO2010/002993. In order to facilitate screening the large library, thelibrary was pooled such that four induced library clones were present ineach well. In order to screen T cells from mice immunized against S.pneumoniae, an aliquot of the pooled library was added toperitoneal-derived macrophages. The macrophages were allowed to bind thetagged S. pneumoniae antigens via the MHC. After 2 hr at 37° C., themacrophages were washed with PBS. The macrophages were then fixed with1% paraformaldehyde for 15 min and washed extensively with PBS. 10⁵ Tcells were added to each well in 200 μL of RP-10 media. The T cells hadpreviously been isolated from mice that had been immunized 2 times withkilled S. pneumoniae bacteria with cholera toxin adjuvant. The assayplates were incubated for 72 hrs at 37° C. The amount of IL-17 in thesupernatant of each well was determined through the use of an IL-17ELISA assay. The threshold for a positive result was set at two standarddeviations above the mean of all samples.

Example 2 Deconvolution of the Positive Murine Pools

A secondary screen was used to determine which antigen(s) out of thefour clones in each well induced the positive response observed in thepooled screen described in Example 1. All the clones in each positivepool were pulsed individually onto peritoneal macrophages in duplicatewells. T cells isolated from immunized mice from the same geneticbackground as the initial screen were used to screen the pulsedmacrophages using the IL-17 assay described in Example 1. Individualantigens that induced an average response in the duplicate wells greaterthan two standard deviations above the mean of negative control sampleswere considered positive responses. The library plasmids present inthese positive clones were sequenced to confirm the identity of theantigen. The antigens SP1574, SP1655, SP2106, SP0148, SP1473, SP0605,SP1177, SP0335, SP0906, SP1828, SP2157, SP1229, SP1128, SP1836, SP1865,SP0904, SP0882, SP0765, SP1634, SP0418, SP1923, SP1313, SP0775, SP0314,SP0912, SP0159, SP0910, SP2148, SP1412, SP0372, SP1304, SP2002, SP0612,SP1988, SP0484, SP0847, SP1527, SP0542, SP0441, SP0350, SP0014, SP1965,SP0117, and SP2108 were confirmed using this method.

Example 3 Antigen Identification and Pooled Human Screens

CD4+ T cells and CD14⁺ monocytes were isolated from peripheral bloodacquired from human donors. The monocytes were differentiated intodendritic cells by culturing them in GM-CSF and IL-4 containing media,essentially as described in Tedder T F and Jansen P J (1997 “Isolationand generation of human dendritic cells.” Current Protocols inImmunology Supp 23: 7.32.1-7.32.16). After five days in culture, thedendritic cells were seeded into 384 well plates. The CD4⁺ T cells werenon-specifically expanded in culture to ensure sufficient quantities.

Each open reading frame predicted in the S. pneumoniae TIGR4 genome wascloned into an expression vector comprising a tag that is able to bepresented by the major histocompatibility complex (MHC). Each constructwas then expressed in E. coli, and full-length expression validated by asurrogate assay that identifies the tag in the context of MHC. In orderto facilitate screening the large library, the library was pooled suchthat four induced library clones were present in each well. In order toscreen the human T cells, an aliquot of the pooled library was added tothe seeded dendritic cells in 384-well plates. After 2 hr at 37° C., thedendritic cells were fixed with 1% paraformaldehyde for 15 min andwashed extensively with phosphate buffer and lysine buffer. 40,000 ofthe CD4⁺ T cells in 70 μL of RP-10 media were added to each well of a384-well plate. The assay plates were incubated for 3 days at 37° C. Theamount of IL-17 in the supernatant of each well was determined throughthe use of an IL-17 ELISA assay. In different iterations of the screen,the threshold for a positive result was set at two standard deviationsabove the mean of all samples, two standard deviations above the mean ofnegative controls, or 1.78 times the median absolution deviation of thedata set. Positive pools were then deconvoluted as described in Example4.

Example 4 Deconvolution of the Positive Human Pools

For all antigens, deconvolution was performed by comparing the resultsof two pool screens. In this method, two different sets of pools wereprepared, so that a polypeptide was with three different polypeptidesbetween the first and second pools. Consequently, it is possible todetermine which polypeptides are antigens by identifying whichpolypeptides are in positive pools in both the first and second sets. Inthis deconvolution method, a pool was identified as positive if it wasat least 1.78 times the median absolution deviation of the data set.

An antigen was identified as a positive hit if it was positive in atleast two repeated secondary screens. The antigens SP2108, SP0641,SP1393, SP0024, SP0641.1, SP1072, SP1384 and SP2032 were identifiedusing the above approach.

Example 5 SP2108, SP0148 and SP1634 Polypeptides

The SP2108 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 7)and SP1634 polypeptide (see Table 2) were formulated as vaccinecompositions using 4 μg of the polypeptide in combination with 1 μgcholera toxin adjuvant (CT). For combinations, 4 μg of each polypeptidewas used. The compositions were administered intranasally to C57BL/6mice three times, one week apart. The subjects were then allowed to restfor 3 weeks, and bled at that time for immunogenicity. For this assay,heparinized whole blood was collected from the retrograde orbital sinus.The total PBMC were stimulated with either killed, unencapsulated wholecell S. pneumoniae (WCC) or a combination of the three polypeptides inround bottomed tubes for three days. The supernatants were thenharvested and evaluated by ELISA for IL-17 levels. Cholera toxin alone(CT) or an unrelated antigen from HSV (003) were used as negativecontrols. Results of the IL-17 immunogenicity assay are shown in FIGS. 1and 2, where the left panels show data in scatter format, and the rightpanels show data as averages with standard deviations. The subjects wereallowed to rest an additional 2 weeks, at which time they werechallenged with intranasal administration of live, encapsulated S.pneumoniae. The subjects were sacrificed a week later, and the number ofcolony-forming units (CFU) was counted from nasal washes. Results of thecolonization assay are shown in FIG. 3.

Example 6 SP0882 and SP0314 Polypeptides

This example used the same protocols as Example 5, except that only twodoses of the vaccine composition were administered. In theseexperiments, the SP0882 polypeptide (SEQ ID NO: 2) and SP0314polypeptides (see Table 2) were tested in parallel with two of the threepolypeptides tested in Example 5. Results of the IL-17 immunogenicityassay are shown in FIGS. 4 and 5. Results of the colonization assay areshown in FIG. 6.

Example 7 SP1072, SP0641N, and SP0024 Polypeptides

This example used a protocol similar to that of Example 5, except thattwo doses of the vaccine compositions were administered, one week apart.Vaccine compositions comprised the polypeptides SP1072 (SEQ ID NO: 8),SP0641N (SEQ ID NO: 13) or SP0024 (SEQ ID NO: 1), and cholera toxinadjuvant (CT). Four weeks after the last immunization, the mice werechallenged intranasally with live type 6B S. pneumoniae. One week laterthe bacterial burden was assessed in each mouse by plating a nasallavage on selective media and counting resultant CFU. The number of CFUisolated from each mouse is plotted for each immunized cohort. Theresults of this colonization assay are shown in FIG. 7. Statisticallysignificant results are indicated in the figure (*=p-value<0.05).

Example 8 SP0148, SP0314, SP0882, and SP2108 Polypeptides Tested in theBALB/c Mouse

To determine whether similar immune responses were seen across differentmouse genotypes, vaccine compositions were administered to BALB/c mice.Vaccine compositions comprised the polypeptides SP0148 (SEQ ID NO: 2),SP0314 (see Table 2), SP0882 (SEQ ID NO: 2) or SP2108 (SEQ ID NO: 9),and cholera toxin adjuvant (CT). Using a protocol similar to that ofExample 5, the mice were immunized, challenged intranasally with S.pneumoniae, and the number of CFU was recorded. The results of thiscolonization experiment are shown in FIG. 8.

Example 9 SP1912, SP2108 and SP0148 Polypeptides IL-17A ImmunogenicityAssay

The polypeptides SP1912 (SEQ ID NO: 265), SP2108 (SEQ ID NO: 9) orSP0148 (SEQ ID NO: 7) were formulated as vaccine compositions withcholera toxin adjuvant (CT). The vaccine compositions were administeredto mice two times, one week apart. The positive control was killed,unencapsulated whole cell S. pneumoniae+CT (WCB), and the negativecontrols were CT alone or recombinant proteins without CT (with theexception of SP1912). Three weeks after the last immunization,peripheral blood was collected from the retroorbital sinus and evaluatedin a whole blood assay. Briefly, the heparizined whole blood was dilutedin media and then cultured in duplicate with A) the protein ofimmunization, or B) the whole cell vaccine for six days. Thesupernatants were harvested and IL-17A levels measured by ELISA. Resultsof the IL-17A immunogenicity assay are shown in FIG. 9. Each symbol inthe graph represents responses from individual mice, and the lineindicates the median response of the group.

Example 10 SP1912, SP2108 and SP0148 Polypeptides Colonization Assay

Animals were immunized with vaccine formulations comprising thepolypeptides SP1912 (SEQ ID NO: 265), SP2108 (SEQ ID NO: 9) or SP0148(SEQ ID NO: 7) and cholera toxin adjuvant (CT) as described in Example9, and then challenged intranasally with 10⁷ live type 6B S. pneumoniaefour weeks after the last immunization (and one week after retroorbitalblood collection). Seven days after challenge, animals were euthanizedand the nasopharyngeal cavities lavaged and cultured on permissive mediato evaluate the S. pneumoniae titers. Results are shown in FIG. 10 asthe colony forming units of bacteria (CFU) per lavage. Each symbolrepresents a titer from an individual mouse response, and the horizontalline represents the median of the group. (***=p-value<0.05).

Example 11 SP1912 Polypeptide Aspiration Challenge (Sepsis Assay)

Polypeptide SP1912 was evaluated for its ability to protect mice fromsepsis. Groups of ten mice were subcutaneously immunized three times,two weeks apart with vaccine compositions comprising either the SP1912polypeptide (SEQ ID NO: 265) or pneumolysoid (PdT) adsorbed to alum. Thepositive control was killed, unencapsulated whole cell S.pneumoniae+alum (WCB), and the negative control was alum alone. Threeweeks after the final immunization, blood was collected for evaluationof IL-17A response and antibody levels, and then one week later, themice underwent aspiration challenge with 10⁷ live strain 0603 (type 6B)S. pneumoniae. Animals were monitored for survival for eight days.Results of the aspiration challenge are shown in FIG. 11 as survivalcurves for each immunized group.

Example 12 Pneumolysoid PdT, SP0148 and SP0641N Polypeptides AspirationChallenge (Sepsis Assay)

Polypeptide SP0148 was evaluated for its ability to protect mice fromsepsis when immunized singly or in combination with SP0641N and/orpneumolysoid (PdT). Groups of ten mice were subcutaneously immunizedthree times, two weeks apart with vaccine compositions comprisingpolypeptide SP0148 (SEQ ID NO: 7), singly or in combination withpolypeptide SP0641N (SEQ ID NO: 13) and/or PdT, adsorbed to alum. Thepositive control was killed, unencapsulated whole cell S.pneumoniae+alum (WCB), and the negative control was alum alone. Threeweeks after the final immunization, blood was collected for evaluationof IL-17 and antibody, and then one week later, the mice underwentaspiration challenge with 10⁷ live strain 0603 (type 6B) S. pneumoniae.Animals were monitored for survival for eight days. The data are shownin FIG. 12 as survival curves for each immunized group.

Example 13 SP1912, SP2108 and SP0148 Polypeptides Colonization Assay

Additional studies were performed essentially as described in Example10, for a total of four separate studies. Briefly, animals wereimmunized with vaccine formulations comprising the polypeptides SP1912(SEQ ID NO: 265), SP2108 (SEQ ID NO: 9), SP0148 (SEQ ID NO: 7), oradditionally SP2108 plus SP0148, and cholera toxin adjuvant (CT) asdescribed in Example 9. Control animals were immunized with killed,unencapsulated whole cell S. pneumoniae plus CT (WCB), or CT alone.Immunized animals were challenged intranasally with 10⁷ live type 6B S.pneumoniae four weeks after the last immunization. Seven days afterchallenge, animals were euthanized and the nasopharyngeal cavitieslavaged and cultured on permissive media to evaluate the S. pneumoniaetiters. Pooled results of four studies are shown in FIG. 13 as thecolony forming units of bacteria (CFU) per lavage. Each symbolrepresents a titer from an individual mouse response, and the horizontalline represents the median of the group. (***=p-value<0.05). N indicatesthe total number of animals evaluated. Percentages refer to the numberof animals protected from colonization.

Example 14 SP1912 and SP0148 Polypeptides IL-17A Immunogenicity Assay

Groups of ten mice were subcutaneously immunized twice, two weeks apartwith vaccine compositions comprising either SP1912 polypeptide (SEQ IDNO: 265), SP0148 polypeptide (SEQ ID NO: 7), or both adsorbed to alum.Control animals were immunized with alum alone. Three weeks after thelast immunization, heparinized blood was collected by cardiac punctureand evaluated for IL-17A levels in a whole blood assay. Briefly, theheparizined whole blood was diluted in media and then cultured for sixdays with the protein(s) of immunization. The supernatants wereharvested and IL-17A levels measured by ELISA. Results of the IL-17Aimmunogenicity assay are shown in FIG. 14. Each symbol in the graphrepresents responses from individual mice, and the line indicates themedian response of the group.

Example 15 SP1912 and SP0148 Polypeptides Colonization Assay

Animals were subcutaneously immunized three times, two weeks apart withvaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 7)at different doses plus and minus SP1912 (SEQ ID NO: 265), adsorbed toalum. Control animals were immunized with killed, unencapsulated wholecell S. pneumoniae plus alum (WCV), or alum alone. Immunized animalswere challenged intranasally with 10⁷ live type 6B S. pneumoniae fourweeks after the last immunization. Seven days after challenge, animalswere euthanized and the nasopharyngeal cavities lavaged and cultured onpermissive media to evaluate the S. pneumoniae titers. Results are shownin FIG. 15 as the colony forming units of bacteria (CFU) per lavage.Each symbol represents a titer from an individual mouse response, andthe horizontal line represents the median of the group. The number ofanimals protected from colonization out of the number of animals in thegroup is indicated at the top of the figure.

Example 16 SP1912, SP0148, and SP2108 Polypeptides Colonization Assay

In two separate studies, animals were subcutaneously immunized threetimes, two weeks apart with vaccine formulations comprising thepolypeptides SP0148 (SEQ ID NO: 7) and SP0148 plus SP1912 (SEQ ID NO:265), or additionally with SP2108 (SEQ ID NO: 9), SP2108 plus SP0148,and SP2108 plus SP1912, adsorbed to alum. Control animals were immunizedwith killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), oralum alone. Immunized animals were challenged intranasally with 10⁷ livetype 6B S. pneumoniae four weeks after the last immunization. Seven daysafter challenge, animals were euthanized and the nasopharyngeal cavitieslavaged and cultured on permissive media to evaluate the S. pneumoniaetiters. Pooled results of the two studies are shown in FIG. 16 as thecolony forming units of bacteria (CFU) per lavage. Each symbolrepresents a titer from an individual mouse response, and the horizontalline represents the median of the group. The number of animals protectedfrom colonization out of the number of animals in the group andcorresponding percentage of animals protected from colonization areindicated at the top of the figure. (*p<0.05, **p<0.01, ***p<0.001Dunn's Multiple Comparison Test compared to Alum control)

Example 17 Pneumolysoid L460D, PspA derivative PR+NPB, SP1912, SP0148,and SP2108 Polypeptides Colonization Assay

Animals were subcutaneously immunized three times, two weeks apart withvaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 7),SP2108 (SEQ ID NO: 9), SP0148 plus SP2108, and SP0148 plus SP2108 incombination with SP1912 (SEQ ID NO: 265) or known S. pneumoniae antigensL460D plus PR+NPD, adsorbed to alum. Two separate studies wereconducted. Control animals were immunized with alum alone. Immunizedanimals were challenged intranasally with 10⁷ live type 6B S. pneumoniaefour weeks after the last immunization. Seven days after challenge,animals were euthanized and the nasopharyngeal cavities lavaged andcultured on permissive media to evaluate the S. pneumoniae titers.Results of the second study are shown in FIG. 17 as the colony formingunits of bacteria (CFU) per lavage. Each symbol represents a titer froman individual mouse response, and the horizontal line represents themedian of the group. The number of animals protected from colonizationout of the number of animals in the group is indicated at the top of thefigure.

The chart below shows the absolute number and corresponding percentageof animals protected from colonization in the four studies described inExamples 16 and 17.

# not colonized/ % not 1 2 3 4 total colonized Alum 0/9 1/10 2/10 4/10 7/39 18% WCB 9/9 9/10 9/10 9/10 36/39 92% 0148  8/18 4/10 5/9  1/1018/47 38% 2108 8/10 6/10 4/10 18/30 60% 1912 4/10  4/10 40% 0148 + 21086/10 3/10 6/10 15/30 50% 0148 + 1912 13/18 3/10 16/28 57% 2108 + 19123/10  3/10 30% 0148 + 2108 + 8/9  8/10 6/10 22/29 76% 1912 0148 + 2108 +2/10 6/10  8/20 40% L460D + PR + NPB

Example 18 PspA, SP0148 and SP2108 Passive Antibody Transfer andAspiration Challenge Sepsis Assay

Groups of ten mice were injected with monoclonal antibodies specific forPspA, heat-inactivated rabbit sera specific for SP0148, SP2108, orcombinations of these. Antibody and antisera concentrations and totalinjection volumes were adjusted with normal rabbit serum (NRS) and PBS.Control animals were injected with NRS, or serum against killed,unencapsulated whole cell S. pneumoniae (WCB). One day after injection,the mice underwent aspiration challenge with 10⁶ live S. pneumoniae typeWU-2 (ST-3). Animals were monitored for survival for eight days. Thedata are shown in FIG. 18 as survival curves for each immunized group.FIG. 19 shows the percent of animals protected from sepsis in thestudies described in Examples 12 and 18, as well as two additionalstudies.

SEQUENCE LISTING

In accordance with 37 C.F.R. §1.52(e)(5), a Sequence Listing in the formof a text file (entitled “Sequence_Listing”, created on Apr. 9, 2012,and 188 kilobytes) is incorporated herein by reference in its entirety.

SEQ ID NO: 1 SP0024>gi|14971488|gb|AAK74215.1|conserved hypothetical protein Streptococcuspneumoniae TIGR4MSYFEQFMQANQAYVALHGQLNLPLKPKTRVAIVTCMDSRLHVAQALGLALGDAHILRNAGGRVTEDMIRSLVISQQQMGTREIVVLHHTDCGAQTFENEPFQEYLKEELGVDVSDQDFLPFQDIEESVREDMQLLIESPLIPDDVIISGAIYNVDTGSMTVVEL SEQ ID NO: 2 SP0882>gi|14972356|gb|AAK75009.1|conserved hypothetical protein (Streptococcuspneumoniae TIGR4)MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW SEQ ID NO: 3 SP0882NMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFI SEQ ID NO: 4SP0882 with exogenous signal sequenceMSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKWSEQ ID NO: 5 SP0882N with exogenous signal sequenceMSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFI SEQ ID NO: 6 SP0148 lacking signal sequenceMCSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKE SEQ ID NO: 7SP0148 including signal sequence (277 amino acids with N-terminal E)MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKE SEQ ID NO: 8 SP1072>gi|14972547|gb|AAK75185.1|DNA primase Streptococcus pneumoniae TIGR4MVDKQVIEEIKNNANIVEVIGDVISLQKAGRNYLGLCPFHGEKTPSFNVVEDKQFYHCFGCGRSGDVFKFIEEYQGVPFIEAVQILGQRVGIEVEKPLYSEQKSASPHQALYDMHEDAAKFYHAILMTTTMGEEARNYLYQRGLTDEVLKHFWIGLAPPERNYLYQRLSDQYREEDLLDSGLFYLSDANQFVDTFHNRIMFPLTNDQGKVIAFSGRIWQKTDSQTSKYKNSRSTAIFNKSYELYHMDRAKRSSGKASEIYLMEGFMDVIAAYRAGIENAVASMGTALSREHVEHLKRLTKKLVLVYDGDKAGQAATLKALDEIGDMPVQIVSMPDNLDPDEYLQKNGPEDLAYLLTKTRISPIEFYIHQYKPENSENLQAQIEFLEKIAPLIVQEKSIAAQNSYIHILADSLASFDYTQIEQIVNESRQVQRQNRMEGISRPTPITMPVTKQLSAIMRAEAHLLYRMMESPLVLNDYRLREDFAFATPEFQVLYDLLGQYGNLPPEVLAEQTEEVERAWYQVLAQDLPAEISPQELSEVEMTRNKALLNQDNMRIKKKVQEASHVGDTDTALEELERLISQKRRME SEQ ID NO: 9SP2108 including signal sequence >gi|14973620|gb|AAK76167.1|maltose/maltodextrin ABC transporter,maltose/maltodextrin-binding protein (Streptococcus pneumoniae TIGR4)MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE SEQ ID NO: 10SP2108 lacking signal sequenceMCGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE SEQ ID NO: 11 SP0641MMSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQGAGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKLDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEALNSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGVIQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNTNKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQFEPIAEGQYFYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEHPEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFTKIHRIKFANQADEKGMISYYLVDPDQDSSKYQ SEQ ID NO: 12 SP0641>gi|14972117|gb|AAK74791.1|serine protease, subtilase family [Streptococcuspneumoniae TIGR4]MKKSTVLSLTTAAVILAAYAPNEVVLADTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAKIKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDNLDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDAKASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDGTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGYEADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDTDKELYKEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYGYMSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQGAGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKLDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEALNSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGVIQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNTNKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQFEPIAEGQYFYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEHPEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFTKIHRIKFANQADEKGMISYYLVDPDQDSSKYQKLGEIAESKFKNLGNGKEGSLKKDTTGVEHHHQENEESIKEKSSFTIDRNISTIRDFENKDLKKLIKKKFREVDDFTSETGKRMEEYDYKYDDKGNIIAYDDGTDLEYETEKLDEIKSKIYGVLSPSKDGHFEILGKISNVSKNAKVYYGNNYKSIEIKATKYDFHSKTMTFDLYANINDIVDGLAFAGDMRLFVKDNDQKKAEIKIRMPEKIKETKSEYPYVSSYGNVIELGEGDLSKNKPDNLTKMESGKIYSDSEKQQYLLKDNIILRKGYALKVTTYNPGKTDMLEGNGVYSKEDIAKIQKANPNLRALSETTIYADSRNVEDGRSTQSVLMSALDGFNIIRYQVFTFKMNDKGEAIDKDGNLVTDSSKLVLFGKDDKEYTGEDKFNVEAIKEDGSMLFIDTKPVNLSMDKNYFNPSKSNKIYVRNPEFYLRGKISDKGGFNWELRVNESVVDNYLIYGDLHIDNTRDFNIKLNVKDGDIMDWGMKDYKANGFPDKVTDMDGNVYLQTGYSDLNAKAVGVHYQFLYDNVKPEVNIDPKGNTSIEYADGKSVVFNINDKRNNGFDGEIQEQHIYINGKEYTSFNDIKQIIDKTLNIKIVVKDFARNTTVKEFILNKDTGEVSELKPHRVTVTIQNGKEMSSTIVSEEDFILPVYKGELEKGYQFDGWEISGFEGKKDAGYVINLSKDTFIKPVFKKIEEKKEEENKPTFDVSKKKDNPQVNHSQLNESHRKEDLQREEHSQKSDSTKDVTATVLDKNNISSKSTTNNPNKLPKTGTASGAQTLLAAGIMFIVGIFLGLKKKNQDSEQ ID NO: 13 SP0641NMVVLADTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAKIKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDNLDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDAKASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDGTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGYEADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDTDKELYKEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG SEQ ID NO: 14SP0882 consensusMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFI                                                     YIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAE  Y               H                 E          E FVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQ                                           EKEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVH      I         H                                        RLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW SEQ ID NO: 15 SP0882N consensusMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFI                                                     YIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAE  Y               H                 E          E  FVMEVVKPFISEQ ID NO: 16 SP0882 consensus with exogenous signal sequenceMSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYE     T  T     V          IKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNE                   Y       Y               HYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNIT E          EQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLM        EK                     I         HDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW                      R SEQ ID NO: 17SP0882N consensus with exogenous signal sequenceMSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYE     T  T     V          IKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNE                   Y       Y               HYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFI  E          E SEQ ID NO: 18SP0148 consensus lacking signal sequenceMCSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKYDVKFE          Q            S  R  N                         N XKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDD                      I                           EIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADLQQIMVRLSDGQFDYKIFDKIG                                      FVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQ           Y      S FFGDTYLPAEADIK(E) SEQ ID NO: 19SP0148 consensus including signal sequenceMKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELT             G     L            Q            S  R  N  GYEIEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAP                 N X                        IIAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKAD            ELQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKF                                Y      SSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIK(E) SEQ ID NO: 20SP2108 consensus lacking signal sequenceMCGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLD                 A                             IKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGA             I        X                T PAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGL                                                        ALAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQE     G         P           A     X                      HGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKNLEASQK                                                         AFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSA      S     A                                  SVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE SEQ ID NO: 21SP2108 consensus including signal sequenceMSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVA     T  T     V                         AKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLS          I                         I        XDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAF   TAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWY                   A        G         P           A     XEKWPKGMQDTEGAGNLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEY                   HAAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDE                    A        S     ALTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQK           SFGE SEQ ID NO: 22 SP1634>gi|14973124|gb|AAK75714.1|hypothetical protein SP_1634 Streptococcuspneumoniae TIGR4MANIFDYLKDVAYDSYYDLPLNELDILTLIEITYLSFDNLVSTLPQRLLDLAPQVPRDPTMLTSKNRLQLLDELAQHKRFKNCKLSHFINDIDPELQKQFAAMTYRVSLDTYLIVFRGTDDSIIGWKEDFHLTYMKEIPAQKHALRYLKNFFAHHPKQKVILAGHSKGGNLAIYAASQIEQSLQNQITAVYTFDAPGLHQELTQTAGYQRIMDRSKIFIPQGSIIGMMLEIPAHQIIVQSTALGGIAQHDTFSWQIEDKHFVQLDKTNSDSQQVDTTFKEWVATVPDEELQLYFDLFFGTILDAGISSINDLASLKALEYIHHLFVQAQSLTPEERETLGRLTQLLIDTRYQAWKNR SEQ ID NO: 23 SP0314>gi|14971788|gb|AAK74491.1|hyaluronidase Streptococcus pneumoniaeTIGR4MQTKTKKLIVSLSSLVLSGFLLNHYMTIGAEETTTNTIQQSQKEVQYQQRDTKNLVENGDFGQTEDGSSPWTGSKAQGWSAWVDQKNSADASTRVIEAKDGAITISSHEKLRAALHRMVPIEAKKKYKLRFKIKTDNKIGIAKVRIIEESGKDKRLWNSATTSGTKDWQTIEADYSPTLDVDKIKLELFYETGTGTVSFKDIELVEVADQLSEDSQTDKQLEEKIDLPIGKKHVFSLADYTYKVENPDVASVKNGILEPLKEGTTNVIVSKDGKEVKKIPLKILASVKDAYTDRLDDWNGIIAGNQYYDSKNEQMAKLNQELEGKVADSLSSISSQADRTYLWEKFSNYKTSANLTATYRKLEEMAKQVTNPSSRYYQDETVVRTVRDSMEWMHKHVYNSEKSIVGNWWDYEIGTPRAINNTLSLMKEYFSDEEIKKYTDVIEKFVPDPEHFRKTTDNPFKALGGNLVDMGRVKVIAGLLRKDDQEISSTIRSIEQVFKLVDQGEGFYQDGSYIDHTNVAYTGAYGNVLIDGLSQLLPVIQKTKNPIDKDKMQTMYHWIDKSFAPLLVNGELMDMSRGRSISRANSEGHVAAVEVLRGIHRIADMSEGETKQCLQSLVKTIVQSDSYYDVFKNLKTYKDISLMQSLLSDAGVASVPRPSYLSAFNKMDKTAMYNAEKGFGFGLSLFSSRTLNYEHMNKENKRGWYTSDGMFYLYNGDLSHYSDGYWPTVNPYKMPGTTETDAKRADSDTGKVLPSAFVGTSKLDDANATATMDFTNWNQTLTAHKSWFMLKDKIAFLGSNIQNTSTDTAATTIDQRKLESGNPYKVYVNDKEASLTEQEKDYPETQSVFLESFDSKKNIGYFFFKKSSISMSKALQKGAWKDINEGQSDKEVENEFLTISQAHKQNRDSYGYMLIPNVDRATFNQMIKELESSLIENNETLQSVYDAKQGVWGIVKYDDSVSTISNQFQVLKRGVYTIRKEGDEYKIAYYNPETQESAPDQEVFKKLEQAAQPQVQNSKEKEKSEEEKNHSDQKNLPQTGEGQSILASLGFLLLGAFYLFRRGKNNSEQ ID NO: 24 SP0882N DNAATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGTGTACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGGCAAAATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCGGATATCAGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAGTTCCAAGAATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTGGTCAAGCCTTTTATC SEQ ID NO: 25 SP0882 with exogenous signal sequence (nucleotides)ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGCATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGTGTACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGGCAAAATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCGGATATCAGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAGTTCCAAGAATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTGGTCAAGCCTTTTATCGATGAGACCTATCGTACAAAAGCAGACTGCCAGCATACGGCTATGATTGGTTCCTCACTAGGAGGCAATATTACCCAGTTTATCGGTTTGGAATACCAAGACCAAATTGGTTGCTTGGGCGTTTTTTCATCTGCAAACTGGCTCCACCAAGAAGCCTTTAACCGCTATTTCGAGTGCCAGAAACTATCGCCTGACCAGCGCATCTTCATCTATGTAGGAACAGAAGAAGCAGATGATACAGACAAGACCTTGATGGATGGCAATATCAAACAAGCCTATATCGACTCGTCGCTTTGCTATTACCATGATTTGATAGCAGGGGGAGTACATCTGGATAATCTTGTGCTAAAAGTTCAGTCTGGTGCCATCCATAGTGAAATCCCTTGGTCAGAAAATCTACCAGATTGTCTGAGATTTTTTGCAGAAAAATGGTAA SEQ ID NO: 26SP0882N with exogenous signal sequence (nucleotides)ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGCATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGTGTACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGGCAAAATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCGGATATCAGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAGTTCCAAGAATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTGGTCAAGCCTTTTATC SEQ ID NO: 27 SP0148 lacking signal sequence (nucleotides)ATGTGCTCAGGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAAGAGTAASEQ ID NO: 28 SP0148 including signal sequence (nucleotides)ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCAGGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAAGAGTAASEQ ID NO: 29 SP2108 lacking signal sequence (nucleotides)ATGTGCGGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAGGGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACTGGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCTCCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACAGACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTTGTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGATAGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACATATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCAAACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACAGAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAAGCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATATGCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTAGACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCTCGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTGCCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAAGATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAATAA SEQ ID NO: 30SP0641M (nucleotides)ATGTCAGGAACTAGTATGGCGACTCCAATCGTGGCAGCTTCTACTGTTTTGATTAGACCGAAATTAAAGGAAATGCTTGAAAGACCTGTATTGAAAAATCTTAAGGGAGATGACAAAATAGATCTTACAAGTCTTACAAAAATTGCCCTACAAAATACTGCGCGACCTATGATGGATGCAACTTCTTGGAAAGAAAAAAGTCAATACTTTGCATCACCTAGACAACAGGGAGCAGGCCTAATTAATGTGGCCAATGCTTTGAGAAATGAAGTTGTAGCAACTTTCAAAAACACTGATTCTAAAGGTTTGGTAAACTCATATGGTTCCATTTCTCTTAAAGAAATAAAAGGTGATAAAAAATACTTTACAATCAAGCTTCACAATACATCAAACAGACCTTTGACTTTTAAAGTTTCAGCATCAGCGATAACTACAGATTCTCTAACTGACAGATTAAAACTTGATGAAACATATAAAGATGAAAAATCTCCAGATGGTAAGCAAATTGTTCCAGAAATTCACCCAGAAAAAGTCAAAGGAGCAAATATCACATTTGAGCATGATACTTTCACTATAGGCGCAAATTCTAGCTTTGATTTGAATGCGGTTATAAATGTTGGAGAGGCCAAAAACAAAAATAAATTTGTAGAATCATTTATTCATTTTGAGTCAGTGGAAGAAATGGAAGCTCTAAACTCCAACGGGAAGAAAATAAACTTCCAACCTTCTTTGTCGATGCCTCTAATGGGATTTGCTGGGAATTGGAACCACGAACCAATCCTTGATAAATGGGCTTGGGAAGAAGGGTCAAGATCAAAAACACTGGGAGGTTATGATGATGATGGTAAACCGAAAATTCCAGGAACCTTAAATAAGGGAATTGGTGGAGAACATGGTATAGATAAATTTAATCCAGCAGGAGTTATACAAAATAGAAAAGATAAAAATACAACATCCCTGGATCAAAATCCAGAATTATTTGCTTTCAATAACGAAGGGATCAACGCTCCATCATCAAGTGGTTCTAAGATTGCTAACATTTATCCTTTAGATTCAAATGGAAATCCTCAAGATGCTCAACTTGAAAGAGGATTAACACCTTCTCCACTTGTATTAAGAAGTGCAGAAGAAGGATTGATTTCAATAGTAAATACAAATAAAGAGGGAGAAAATCAAAGAGACTTAAAAGTCATTTCGAGAGAACACTTTATTAGAGGAATTTTAAATTCTAAAAGCAATGATGCAAAGGGAATCAAATCATCTAAACTAAAAGTTTGGGGTGACTTGAAGTGGGATGGACTCATCTATAATCCTAGAGGTAGAGAAGAAAATGCACCAGAAAGTAAGGATAATCAAGATCCTGCTACTAAGATAAGAGGTCAATTTGAACCGATTGCGGAAGGTCAATATTTCTATAAATTTAAATATAGATTAACTAAAGATTACCCATGGCAGGTTTCCTATATTCCTGTAAAAATTGATAACACCGCCCCTAAGATTGTTTCGGTTGATTTTTCAAATCCTGAAAAAATTAAGTTGATTACAAAGGATACTTATCATAAGGTAAAAGATCAGTATAAGAATGAAACGCTATTTGCGAGAGATCAAAAAGAACATCCTGAAAAATTTGACGAGATTGCGAACGAAGTTTGGTATGCTGGCGCCGCTCTTGTTAATGAAGATGGAGAGGTTGAAAAAAATCTTGAAGTAACTTACGCAGGTGAGGGTCAAGGAAGAAATAGAAAACTTGATAAAGACGGAAATACCATTTATGAAATTAAAGGTGCGGGAGATTTAAGGGGAAAAATCATTGAAGTCATTGCATTAGATGGTTCTAGCAATTTCACAAAGATTCATAGAATTAAATTTGCTAATCAGGCTGATGAAAAGGGGATGATTTCCTATTATCTAGTAGATCCTGATCAAGATTCATCTAAATATCAA SEQ ID NO: 31 SP0641N (nucleotides)ATGGTAGTCTTAGCAGACACATCTAGCTCTGAAGATGCTTTAAACATCTCTGATAAAGAAAAAGTAGCAGAAAATAAAGAGAAACATGAAAATATCCATAGTGCTATGGAAACTTCACAGGATTTTAAAGAGAAGAAAACAGCAGTCATTAAGGAAAAAGAAGTTGTTAGTAAAAATCCTGTGATAGACAATAACACTAGCAATGAAGAAGCAAAAATCAAAGAAGAAAATTCCAATAAATCCCAAGGAGATTATACGGACTCATTTGTGAATAAAAACACAGAAAATCCCAAAAAAGAAGATAAAGTTGTCTATATTGCTGAATTTAAAGATAAAGAATCTGGAGAAAAAGCAATCAAGGAACTATCCAGTCTTAAGAATACAAAAGTTTTATATACTTATGATAGAATTTTTAACGGTAGTGCCATAGAAACAACTCCAGATAACTTGGACAAAATTAAACAAATAGAAGGTATTTCATCGGTTGAAAGGGCACAAAAAGTCCAACCCATGATGAATCATGCCAGAAAGGAAATTGGAGTTGAGGAAGCTATTGATTACCTAAAGTCTATCAATGCTCCGTTTGGGAAAAATTTTGATGGTAGAGGTATGGTCATTTCAAATATCGATACTGGAACAGATTATAGACATAAGGCTATGAGAATCGATGATGATGCCAAAGCCTCAATGAGATTTAAAAAAGAAGACTTAAAAGGCACTGATAAAAATTATTGGTTGAGTGATAAAATCCCTCATGCGTTCAATTATTATAATGGTGGCAAAATCACTGTAGAAAAATATGATGATGGAAGGGATTATTTTGACCCACATGGGATGCATATTGCAGGGATTCTTGCTGGAAATGATACTGAACAAGACATCAAAAACTTTAACGGCATAGATGGAATTGCACCTAATGCACAAATTTTCTCTTACAAAATGTATTCTGACGCAGGATCTGGGTTTGCGGGTGATGAAACAATGTTTCATGCTATTGAAGATTCTATCAAACACAACGTTGATGTTGTTTCGGTATCATCTGGTTTTACAGGAACAGGTCTTGTAGGTGAGAAATATTGGCAAGCTATTCGGGCATTAAGAAAAGCAGGCATTCCAATGGTTGTCGCTACGGGTAACTATGCGACTTCTGCTTCAAGTTCTTCATGGGATTTAGTAGCAAATAATCATCTGAAAATGACCGACACTGGAAATGTAACACGAACTGCAGCACATGAAGATGCGATAGCGGTCGCTTCTGCTAAAAATCAAACAGTTGAGTTTGATAAAGTTAACATAGGTGGAGAAAGTTTTAAATACAGAAATATAGGGGCCTTTTTCGATAAGAGTAAAATCACAACAAATGAAGATGGAACAAAAGCTCCTAGTAAATTAAAATTTGTATATATAGGCAAGGGGCAAGACCAAGATTTGATAGGTTTGGATCTTAGGGGCAAAATTGCAGTAATGGATAGAATTTATACAAAGGATTTAAAAAATGCTTTTAAAAAAGCTATGGATAAGGGTGCACGCGCCATTATGGTTGTAAATACTGTAAATTACTACAATAGAGATAATTGGACAGAGCTTCCAGCTATGGGATATGAAGCGGATGAAGGTACTAAAAGTCAAGTGTTTTCAATTTCAGGAGATGATGGTGTAAAGCTATGGAACATGATTAATCCTGATAAAAAAACTGAAGTCAAAAGAAATAATAAAGAAGATTTTAAAGATAAATTGGAGCAATACTATCCAATTGATATGGAAAGTTTTAATTCCAACAAACCGAATGTAGGTGACGAAAAAGAGATTGACTTTAAGTTTGCACCTGACACAGACAAAGAACTCTATAAAGAAGATATCATCGTTCCAGCAGGATCTACATCTTGGGGGCCAAGAATAGATTTACTTTTAAAACCCGATGTTTCAGCACCTGGTAAAAATATTAAATCCACGCTTAATGTTATTAATGGCAAATCAACTTATGGCSEQ ID NO: 32 HHHHHH SEQ ID NO: 33 MSYYHHHHHH SEQ ID NO: 265 SP1912MNGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVL SEQ ID NO: 266 SP1912LMRYLATLLLSLAVLITAGCKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVL SEQ ID NO: 267 SP0641.1DTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAKIKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDNLDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDAKASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDGTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGYEADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDTDKELYKEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYGYMSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQGAGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKLDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEALNSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGVIQNRKDKNTTSL SEQ ID NO: 268SP1912 consensusMNGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIF                                H          A    L           I          L SEDGRHYTFVYENEDLVYEEEVL         I SEQ ID NO: 269 SP641N consensusMVVLADTSSSEDALNISDKEKVA-----ENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAK—      N     S       VVDKET      KD  N I   K TE    TI EG A       T TK      R                       LIKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDN   D—      H              Q               S    Q  N                G     Q            NAH SA  G        RL                 GLDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDA          T IKASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDG                                                      Q             NTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGYEADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDT                           NDKELYKEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG SEQ ID NO: 270SP641M consensusMSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQG                           K      TAGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKL                                                                 VDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEAL          Y                                            R                ANSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGVS     TD                               K   MEIQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNT        R     D D      Q VH E TNKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQF       K    V             GEPIAEGQYFYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEHPEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFT                                                              S         AKIHRIKFANQADEKGMISYYLVDPDQDSSKYQ         DH               K A   ESEQ ID NO: 271 SP1912 (nucleotides)ATGAATGGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCTACGAAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTTGAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGASEQ ID NO: 272 SP1912L (nucleotides)ATGAGATACCTGGCAACATTGTTGTTATCTCTGGCGGTGTTAATCACCGCCGGGTGCAAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTTGAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGA SEQ ID NO: 273SP0641.1(nucleotides)GACACATCTAGCTCTGAAGATGCTTTAAACATCTCTGATAAAGAAAAAGTAGCAGAAAATAAAGAGAAACATGAAAATATCCATAGTGCTATGGAAACTTCACAGGATTTTAAAGAGAAGAAAACAGCAGTCATTAAGGAAAAAGAAGTTGTTAGTAAAAATCCTGTGATAGACAATAACACTAGCAATGAAGAAGCAAAAATCAAAGAAGAAAATTCCAATAAATCCCAAGGAGATTATACGGACTCATTTGTGAATAAAAACACAGAAAATCCCAAAAAAGAAGATAAAGTTGTCTATATTGCTGAATTTAAAGATAAAGAATCTGGAGAAAAAGCAATCAAGGAACTATCCAGTCTTAAGAATACAAAAGTTTTATATACTTATGATAGAATTTTTAACGGTAGTGCCATAGAAACAACTCCAGATAACTTGGACAAAATTAAACAAATAGAAGGTATTTCATCGGTTGAAAGGGCACAAAAAGTCCAACCCATGATGAATCATGCCAGAAAGGAAATTGGAGTTGAGGAAGCTATTGATTACCTAAAGTCTATCAATGCTCCGTTTGGGAAAAATTTTGATGGTAGAGGTATGGTCATTTCAAATATCGATACTGGAACAGATTATAGACATAAGGCTATGAGAATCGATGATGATGCCAAAGCCTCAATGAGATTTAAAAAAGAAGACTTAAAAGGCACTGATAAAAATTATTGGTTGAGTGATAAAATCCCTCATGCGTTCAATTATTATAATGGTGGCAAAATCACTGTAGAAAAATATGATGATGGAAGGGATTATTTTGACCCACATGGGATGCATATTGCAGGGATTCTTGCTGGAAATGATACTGAACAAGACATCAAAAACTTTAACGGCATAGATGGAATTGCACCTAATGCACAAATTTTCTCTTACAAAATGTATTCTGACGCAGGATCTGGGTTTGCGGGTGATGAAACAATGTTTCATGCTATTGAAGATTCTATCAAACACAACGTTGATGTTGTTTCGGTATCATCTGGTTTTACAGGAACAGGTCTTGTAGGTGAGAAATATTGGCAAGCTATTCGGGCATTAAGAAAAGCAGGCATTCCAATGGTTGTCGCTACGGGTAACTATGCGACTTCTGCTTCAAGTTCTTCATGGGATTTAGTAGCAAATAATCATCTGAAAATGACCGACACTGGAAATGTAACACGAACTGCAGCACATGAAGATGCGATAGCGGTCGCTTCTGCTAAAAATCAAACAGTTGAGTTTGATAAAGTTAACATAGGTGGAGAAAGTTTTAAATACAGAAATATAGGGGCCTTTTTCGATAAGAGTAAAATCACAACAAATGAAGATGGAACAAAAGCTCCTAGTAAATTAAAATTTGTATATATAGGCAAGGGGCAAGACCAAGATTTGATAGGTTTGGATCTTAGGGGCAAAATTGCAGTAATGGATAGAATTTATACAAAGGATTTAAAAAATGCTTTTAAAAAAGCTATGGATAAGGGTGCACGCGCCATTATGGTTGTAAATACTGTAAATTACTACAATAGAGATAATTGGACAGAGCTTCCAGCTATGGGATATGAAGCGGATGAAGGTACTAAAAGTCAAGTGTTTTCAATTTCAGGAGATGATGGTGTAAAGCTATGGAACATGATTAATCCTGATAAAAAAACTGAAGTCAAAAGAAATAATAAAGAAGATTTTAAAGATAAATTGGAGCAATACTATCCAATTGATATGGAAAGTTTTAATTCCAACAAACCGAATGTAGGTGACGAAAAAGAGATTGACTTTAAGTTTGCACCTGACACAGACAAAGAACTCTATAAAGAAGATATCATCGTTCCAGCAGGATCTACATCTTGGGGGCCAAGAATAGATTTACTTTTAAAACCCGATGTTTCAGCACCTGGTAAAAATATTAAATCCACGCTTAATGTTATTAATGGCAAATCAACTTATGGCTATATGTCAGGAACTAGTATGGCGACTCCAATCGTGGCAGCTTCTACTGTTTTGATTAGACCGAAATTAAAGGAAATGCTTGAAAGACCTGTATTGAAAAATCTTAAGGGAGATGACAAAATAGATCTTACAAGTCTTACAAAAATTGCCCTACAAAATACTGCGCGACCTATGATGGATGCAACTTCTTGGAAAGAAAAAAGTCAATACTTTGCATCACCTAGACAACAGGGAGCAGGCCTAATTAATGTGGCCAATGCTTTGAGAAATGAAGTTGTAGCAACTTTCAAAAACACTGATTCTAAAGGTTTGGTAAACTCATATGGTTCCATTTCTCTTAAAGAAATAAAAGGTGATAAAAAATACTTTACAATCAAGCTTCACAATACATCAAACAGACCTTTGACTTTTAAAGTTTCAGCATCAGCGATAACTACAGATTCTCTAACTGACAGATTAAAACTTGATGAAACATATAAAGATGAAAAATCTCCAGATGGTAAGCAAATTGTTCCAGAAATTCACCCAGAAAAAGTCAAAGGAGCAAATATCACATTTGAGCATGATACTTTCACTATAGGCGCAAATTCTAGCTTTGATTTGAATGCGGTTATAAATGTTGGAGAGGCCAAAAACAAAAATAAATTTGTAGAATCATTTATTCATTTTGAGTCAGTGGAAGAAATGGAAGCTCTAAACTCCAACGGGAAGAAAATAAACTTCCAACCTTCTTTGTCGATGCCTCTAATGGGATTTGCTGGGAATTGGAACCACGAACCAATCCTTGATAAATGGGCTTGGGAAGAAGGGTCAAGATCAAAAACACTGGGAGGTTATGATGATGATGGTAAACCGAAAATTCCAGGAACCTTAAATAAGGGAATTGGTGGAGAACATGGTATAGATAAATTTAATCCAGCAGGAGTTATACAAAATAGAAAAGATAAAAATACAACATCCCTG SEQ ID NO: 274 Canonical lipobox motif[LIVMFESTAGPC]-[LVIAMFTG]-[IVMSTAGCP]-[AGS]-C SEQ ID NO: 275SP2108 signal sequence MSSKFMKSAAVLGTATLASLLLVAC SEQ ID NO: 276E. coli RlpB signal sequence MRYLATLLLSLAVLITAG[C] SEQ ID NO: 301Immunogenic PspA/PspC polypeptides including the coiled-coil structure (PR +NPB) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMWCRQACGRTRAPPPPPLRSGC SEQ ID NO: 302Immunogenic PspA/PspC polypeptides including the coiled-coil structure (PRonly) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMWCRQACGRTRAPPPPPLRSG SEQ ID NO: 303Immunogenic PspA/PspC polypeptides lacking the coiled-coil structure (PR +NPB) DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMW SEQ ID NO: 304Immunogenic PspA/PspC polypeptides lacking the coiled-coil structure (PRonly) DLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMW SEQ ID NO: 305Immunogenic PspA/PspC polypeptides lacking the coiled-coil structure (PR +NPB) MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPKASEQ ID NO: 306 Non-proline Block (NPB) EKSADQQAEEDYARRSEEEYNRLTQQQSEQ ID NO: 307 Non-proline Block (NPB) DQQAEEDYARRSEEEYNRLTQQQSEQ ID NO: 308 Non-proline Block (NPB) MEKSADQQAEEDYARRSEEEYNRLTQQQSEQ ID NO: 309 Amino-terminal boundary to the PR-region DLKKAVNESEQ ID NO: 310 Carboxy-terminal boundary to the PR-region(K/G)TGW(K/G)QENGMW

We claim:
 1. A vaccine formulation comprising: a pharmaceuticallyacceptable carrier; an effective amount of an adjuvant; a first isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 265, 266,or 268, or comprising the amino acid sequence of SEQ ID NO:265, 266, or268 lacking 1-3 amino acids from the N-terminus, C-terminus, or both;and a second isolated polypeptide comprising the amino acid sequence ofSEQ ID NO:9 or 10, or comprising the amino acid sequence of SEQ ID NO:9or 10 lacking 1-3 amino acids from the N-terminus, C-terminus, or both.2. The vaccine formulation of claim 1, further comprising a thirdisolated polypeptide comprising the amino acid sequence of SEQ ID NO:6or 7, or comprising the amino acid sequence of SEQ ID NO:6 or 7 lacking1-3 amino acids from the N-terminus, C-terminus, or both.
 3. The vaccineformulation of claim 2, wherein the third polypeptide consists of theamino acid sequence of SEQ ID NO:6.
 4. The vaccine formulation of claim2, wherein the third polypeptide consists of the amino acid sequence ofSEQ ID NO:7.
 5. A vaccine formulation comprising: a pharmaceuticallyacceptable carrier; an effective amount of an adjuvant; a first isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:265, 266, or268, or comprising the amino acid sequence of SEQ ID NO:265, 266, or 268lacking 1-3 amino acids from the N-terminus, C-terminus, or both; and asecond isolated polypeptide comprising the amino acid sequence of SEQ IDNO:6 or 7, or comprising the amino acid sequence of SEQ ID NO:6 or 7lacking 1-3 amino acids from the N-terminus, C-terminus, or both.
 6. Thevaccine formulation of claim 1 or 5, wherein the first polypeptideconsists of the amino acid sequence of SEQ ID NO:265.
 7. The vaccineformulation of claim 1 or 5, wherein the first polypeptide consists ofthe amino acid sequence of SEQ ID NO:266.
 8. The vaccine formulation ofclaim 1 or 5, wherein the first polypeptide consists of the amino acidsequence of SEQ ID NO:268.
 9. The vaccine formulation of claim 1,wherein the second polypeptide consists of the amino acid sequence ofSEQ ID NO:9.
 10. The vaccine formulation of claim 1 or 5, wherein one ormore polypeptides are conjugated to an immunogenic carrier.
 11. Thevaccine formulation of claim 1 or 5, which comprises at least onelipidated polypeptide.
 12. The vaccine formulation of claim 5, whereinthe second polypeptide consists of the amino acid sequence of SEQ IDNO:6.
 13. The vaccine formulation of claim 5, wherein the secondpolypeptide consists of the amino acid sequence of SEQ ID NO:7.
 14. Avaccine formulation comprising: a pharmaceutically acceptable carrier;an effective amount of an adjuvant; a first isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 265, 266, or 268 orcomprising the amino acid sequence of SEQ ID NO:265, 266, or 268 lacking1-3 amino acids from the N-terminus, C-terminus, or both; a secondisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 6or comprising the amino acid sequence of SEQ ID NO:6 lacking 1-3 aminoacids from the N-terminus, C-terminus, or both; and a third isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:7 orcomprising the amino acid sequence of SEQ ID NO:7 lacking 1-3 aminoacids from the N-terminus, C-terminus, or both.
 15. The vaccineformulation of claim 14, wherein the second polypeptide is lipidated.16. The vaccine formulation of claim 14 or 15, further comprising one orboth of: a fourth isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 9 or comprising the amino acid sequence of SEQ IDNO: 9 lacking 1-3 amino acids from the N-terminus, C-terminus, or both;and a fifth isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 10 or comprising the amino acid sequence of SEQ ID NO: 10lacking 1-3 amino acids from the N-terminus, C-terminus, or both. 17.The vaccine formulation of claim 16, wherein the fifth polypeptide islipidated.
 18. A vaccine formulation comprising: a pharmaceuticallyacceptable carrier; an effective amount of an adjuvant; a first isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 265, 266,or 268 or comprising the amino acid sequence of SEQ ID NO: 265, 266, or268 lacking 1-3 amino acids from the N-terminus, C-terminus, or both; asecond isolated polypeptide comprising the amino acid sequence of SEQ IDNO: 9 or comprising the amino acid sequence of SEQ ID NO: 9 lacking 1-3amino acids from the N-terminus, C-terminus, or both; and a thirdisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10or comprising the amino acid sequence of SEQ ID NO: 10 lacking 1-3 aminoacids from the N-terminus, C-terminus, or both.
 19. The vaccineformulation of claim 18, wherein the third polypeptide is lipidated. 20.A vaccine formulation comprising: a pharmaceutically acceptable carrier;an effective amount of an adjuvant; a first isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 265 or comprising theamino acid sequence of SEQ ID NO:265 lacking 1-3 amino acids from theN-terminus, C-terminus, or both; a second isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 6 or comprising theamino acid sequence of SEQ ID NO:6 lacking 1-3 amino acids from theN-terminus, C-terminus, or both; a third isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:7 or comprising the amino acidsequence of SEQ ID NO:7 lacking 1-3 amino acids from the N-terminus,C-terminus, or both; a fourth isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 9 or comprising the amino acid sequence ofSEQ ID NO: 9 lacking 1-3 amino acids from the N-terminus, C-terminus, orboth; and a fifth isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 10 or comprising the amino acid sequence of SEQID NO: 10 lacking 1-3 amino acids from the N-terminus, C-terminus, orboth.
 21. The vaccine formulation of claim 20, wherein the adjuvant isalum.
 22. The vaccine formulation of claim 20, wherein the vaccineformulation comprises 1-1000 μg of each polypeptide.
 23. The vaccineformulation of any one of claim 1, 5, 14, 18, or 20, wherein uponadministration to a subject, the vaccine formulation induces an immuneresponse against S. pneumoniae.
 24. A pharmaceutical compositioncomprising: a pharmaceutically acceptable carrier; an effective amountof an adjuvant; a first isolated polypeptide comprising an amino acidsequence at least 98% identical to the amino acid sequence of SEQ IDNO:265, 266, or 268, or comprising an amino acid sequence at least 98%identical to the amino acid sequence of SEQ ID NO:265, 266, or 268lacking 1-3 amino acids from the N-terminus, C-terminus, or both; and asecond isolated polypeptide comprising an amino acid sequence at least98% identical to the amino acid sequence of SEQ ID NO:9 or 10, orcomprising an amino acid sequence at least 98% identical to the aminoacid sequence of SEQ ID NO:9 or 10 lacking 1-3 amino acids from theN-terminus, C-terminus, or both.
 25. The pharmaceutical composition ofclaim 24, further comprising a third isolated polypeptide comprising anamino acid sequence at least 98% identical to the amino acid sequence ofSEQ ID NO:6 or 7, or comprising an amino acid sequence at least 98%identical to the amino acid sequence of SEQ ID NO:6 or 7 lacking 1-3amino acids from the N-terminus, C-terminus, or both.
 26. Apharmaceutical composition comprising: a pharmaceutically acceptablecarrier; an effective amount of an adjuvant; a first isolatedpolypeptide comprising an amino acid sequence at least 98% identical tothe amino acid sequence of SEQ ID NO:265, 266, or 268, or comprising anamino acid sequence at least 98% identical to the amino acid sequence ofSEQ ID NO:265, 266, or 268 lacking 1-3 amino acids from the N-terminus,C-terminus, or both; and a second isolated polypeptide comprising anamino acid sequence at least 98% identical to the amino acid sequence ofSEQ ID NO:6 or 7, or comprising an amino acid sequence at least 98%identical to the amino acid sequence of SEQ ID NO:6 or 7 lacking 1-3amino acids from the N-terminus, C-terminus, or both.
 27. Thepharmaceutical composition of 90 or 92, wherein upon administration to asubject, the pharmaceutical composition induces an immune responseagainst S. pneumoniae.
 28. A pharmaceutical composition comprising: apharmaceutically acceptable carrier; an effective amount of an adjuvant;a first polypeptide comprising an amino acid sequence from a strain ofS. pneumoniae that corresponds to the amino acid sequence of SEQ ID NO:265, 266, or 268, or to the amino acid sequence of SEQ ID NO:265, 266,or 268 lacking 1-3 amino acids from the N-terminus, C-terminus, or both;and a second polypeptide comprising an amino acid sequence from a strainof S. pneumoniae that corresponds to the amino acid sequence of SEQ IDNO:9 or 10, or to the amino acid sequence of SEQ ID NO:9 or 10 lacking1-3 amino acids from the N-terminus, C-terminus, or both.
 29. Thepharmaceutical composition of claim 28, further comprising a thirdpolypeptide comprising an amino acid sequence from a strain of S.pneumoniae that corresponds to the amino acid sequence of SEQ ID NO:6 or7, or to the amino acid sequence of SEQ ID NO:6 or 7 lacking 1-3 aminoacids from the N-terminus, C-terminus, or both.